CONTRIBUTIONS FROM THE LABORATORY OF 
THE ROTHAMSTED EXPERIMENTAL STATION 

(LAWES AGRICULTURAL TRUST) 



Reprinted from THE JOURNAL OF AGRICULTURAL SCIENCE, October, 1909. 




CAMBRIDGE 
AT THE UNIVERSITY PRESS 



CONTRIBUTIONS FROM THE LABORATORY OF 
THE ROTHAMSTED EXPERIMENTAL STATION 

(LAWES AGRICULTURAL TRUST) 



Reprinted prom THE JOURNAL OF AGRICULTURAL SCIENCE, October, 1909. 



' i ? 




1 1 * 



mk 



CAMBRIDGE 
AT THE UNIVERSITY PRESS 






By rpAVT — > 
AUG 22 1910 



CONTENTS. 

(All Bights reserved.) 

PAGE 

1. Russell, Edward John, and Hutchinson, Henry Brougham. 
The effect of partial sterilisation of soil on the production 
of plant food. (Four figures in text. Plates VIII, IX) . Ill 

:2. Marr, F. S. Estimation of calcium carbonate in soils . . 155 

•3. Morison, C. G. T. The amount of free lime and the composition 

of the soluble phosphates in basic slag (Two figures in text.) 161 

4. Hutchinson, H. B. ; and Miller, N. K. J. Direct assimilation of 

ammonium salts by plants. (Two figures in text. Plate XIV.) 179 

5. Brenchley, W. E., and Hall, A. D. The development of 

the grain of wheat. (Twenty figures in text. Plate XV.) . 195 



Digitized by the Internet Archive 
in 2010 with funding from 
The Library of Congress 



http://www.archive.org/details/contributionsfroOOroth 



Volume III OCTOBER, 1909 Part II 



THE EFFECT OF PARTIAL STERILISATION OF 
SOIL ON THE PRODUCTION OF PLANT FOOD. 

By EDWARD JOHN RUSSELL, D.Sc. (Lond.), and 
HENRY BROUGHAM HUTCHINSON, Ph.D. 

[Rothamsted Experiment Station.) 

Introduction. 

When soil is partially sterilised, either by heat or by volatile 
antiseptics like carbou disulphide, toluene, etc., it becomes more pro- 
ductive and capable of yielding larger crops. The effect of heat was 
discovered incidentally about 25 years ago by the early soil bacteriolo- 
gists ; the action of carbon disulphide was first noticed somewhat later 
by a vine grower who had used it to kill phylloxera. Both cases have 
since been studied by several investigators, notably Koch 1 and Hiltner 
and Stormer 2 ; a paper was also recently published by one of us 3 in 
which it was shown that the property is a general one, holding for all 
the soils and volatile antiseptics examined and for all the plants, 
excepting those of the leguminous order. Thus when a soil had been 
heated to 95° C. it produced two, three, or sometimes four times as 
much crop as a portion of the soil which had not been heated, whilst 
treatment with volatile antiseptics led to an increase in crop varying 
between 20 and 50 per cent. The treatment had in some way brought 
about a considerable increase in the amount of plant food — nitrogen, 
phosphorus, and potassium — obtainable by the plant ; even more, 
indeed, than might be expected from the weight of the crop, since there 
was an increased percentage of nitrogen and phosphorus in the 
material of plants grown on the treated soils. The results quoted in 

1 Koch, Arbeiten der deutschen Landwirtschaft-Gesellschaft, 1899, Heft 40. 

2 Hiltner and Stormer, Arbeiten der Biolog. Abteilung f. Land- u. Forsticirtschaft, 
1903, Bd. 3, Heft 5. 

3 Darbishire and Russell, Journal of Agricultural Science, 1908, Vol. n. p. 305. Full 
references to the literature of the subject are given in this paper. 

Journ. of Agric. Sci. in 8 



112 Partial Sterilisation of Soil 

the earlier paper were obtained with fertile soils, but we have obtained 
precisely similar results with an exhausted Rothamsted soil. (See 
Plate VIII, Figs. 1 and 2 and Table 1.) 

Several hypotheses have been put forward to account for the 
increased productiveness. It was first supposed that a chemical reaction 
took place between the antiseptic and the soil whereby plant food was 
rendered more available ; this view was soon discarded, but has recently 
been revived by Pickering 1 . Koch suggested a purely physiological 
hypothesis ; the antiseptic was supposed to stimulate the plant roots to 
greater activity in extracting food from the soil. Such an action might 
have gone on in Koch's experiments where the antiseptic was left in 
the soil, but can hardly have taken place in ours, since all the antiseptic 
had been removed before the seeds were sown. Hiltner and Stormer 
attribute the action to the changed bacterial flora. They showed that 
the first effect of the antiseptic is to reduce the number of organisms, 
but when the conditions again became favourable the survivors multiply 
with extraordinary rapidity, and bring about a more intense production 
of nitrogenous plant food in the soil. They supposed that a larger 
amount of atmospheric nitrogen is " fixed," and the complex substances 
thus formed in the bacterial cells are slowly broken down to yield plant 
food. The decomposition processes normally taking place in the soil 
are probably hastened also, whilst the loss of nitrogen by denitrification 
is diminished. Other investigators have also supposed that increased 
nitrogen fixation is the main cause of the increased productiveness ; on 
the other hand Koch 2 maintains that nitrogen fixation is decreased by 
partial sterilisation. Stormer 3 considers that the larger organisms are 
killed and decomposed by the surviving bacteria with production of 
ammonia. The dark green colour of the plants grown on partially 
sterilised soils has generally been regarded as an indication that the 
nitrogenous food stuff in the soil has in some way been increased by 
the treatment. 

Part 1. 

§ 1. We propose to give in this part a short statement of our 
experiments and the conclusions to which they lead, reference being made 
at each step to the paragraph in Part 2, where the full details and figures 

1 S. TJ. Pickering, Journal of Agricultural Science, 1909, Vol. in. p. 411. 

2 Koch, Journal fur Landivirtschaft, 1907, Bd. 55, S. 355. 

3 Stormer, Jahresber. d. Vereinigung filr Angewandte Botanik, 1907, S. 113. 



E. J. Russell and H. B. Hutchinson 



113 



are given. At the outset we may state that the soil employed in the 
experiments was taken from an arable field and contained moderate 
but not large amounts of nitrogen, organic matter, and calcium carbonate 
(§ 14). Partial sterilisation was effected either by heating to 98° C. or 
by addition of 4 per cent, of toluene, which at the end of three days 
was allowed to evaporate by spreading out the soil in a thin layer for 
as long as might be necessary. For convenience this soil is called 
" toluene evaporated " to distinguish it from a third series where the 
toluene was left in during the whole of the experimental period. 



50 • 



cs 40 - 

a 



2 30 



20 - 



10 - 



A^ 



tf>° 



Toluene 8-*""* 



Toluene left in 



Untreated soil 



P-> 



days 



4 8 12 16 20 24 

Curve 1. Amount of Ammonia in variously treated soils (Table 2). 

A fourth series consisted of untreated soils; a few experiments were 
also made with soils heated to 125° C, at which temperature all 
organisms are killed. After treatment the soils were moistened and 
kept for definite periods in bottles stopped with cotton-wool at the 
ordinary laboratory temperature. In these circumstances various 
changes soon set in, and are dealt with below. 

I. The changes taking place in partially sterilised soils. 

§ 2. (a) Ammonia. Curve 1 shows the amount of ammonia found 
in the various soils at stated intervals after the moisture had been 
added. In the untreated soil there is no accumulation of ammonia. 
The " toluene evaporated " soil and the soil heated to 98° C. show 

8—2 



114 



Partial Sterilisation of Soil 



that the treatment has effected a small immediate production of 
ammonia amounting to about 5 parts per million of soil, then little 
further change takes place for a few days. This period of comparative 
inaction is followed by one of rapid change, during which ammonia is 
produced in considerable quantity ; lastly a slow period sets in, and the 
further production of ammonia is now only small. By the end of 
a month about 40 parts of nitrogen per million of soil have been con- 
verted into ammonia. (§ 15, Table 2.) 

The difference between the "toluene evaporated" and the heated 
soils is only one of degree. The acceleration in the rate of formation of 




20 hours 



Curve 2. Amount of ammonia produced from peptone solution after inoculation 
with arable soil (§ 24). 

ammonia is evident at an earlier date in the toluened than in the 
heated soil, but is not maintained for so long, and by the ninth day 
the heated soil already contains more ammonia, a superiority which it 
maintains throughout. 

The production of ammonia is mainly the work of micro- organisms. 
Proof is furnished by the following considerations : 

1. The curves belong to the type associated with bacterial, rather 
than purely chemical change (cf. Curve 2). 

2. Soil which has been heated to 125° C. (at which temperature 
all organisms are killed) behaves altogether differently : after the first 
production of ammonia due to heating there is no further change. 



E. J. Russell and H. B. Hutchinson 115 

3. If the toluene is left in the soil there is only a slow production 
of ammonia and never a rapid rate ; the curve is more nearly linear. 
The action of micro-organisms is here excluded, but enzymes may still 
act. 

4. The rapid period sets in only when the soil is sufficiently moist. 
(Table 2.) 

(b) The production of unstable nitrogen compounds, which may 
be regarded as intermediate products in the general decomposition, 
is also accelerated by partial sterilisation. (§ 17, Table 3.) 

(c) The humus, on the other hand, appears to be but little affected ; 
if anything there is a small increase, rather than a decrease, in the 
amount of humic nitrogen. It does not appear that the ammonia has 
been produced in the partially sterilised soils at the expense of humic 
nitrogen. (§ 19, Table 5.) 

(d) Nitrification. The nitrifying organisms are destroyed by either 
method of partial sterilisation, but there is a very important difference 
between the two cases to which subsequent reference will be made. 
Toluene simply destroys the organisms: if they are again introduced 
after the toluene has been removed they at once begin to act. Heat 
not only destroys the organisms but brings about some change whereby 
the soil is rendered unsuitable for their development ; they now no 
longer act even w r hen re-introduced into the soil. (§ 31.) It appears 
that an inhibitory substance is formed by heat. References to Table 2 
show that the untreated soil gains in nitrate whilst the toluened and 
heated soil do not. 

(e) The change in the total amount of nitrogen is not great, even 
over a long period. There appears to be a small net loss from the 
partially sterilised soils as compared with the untreated soil : whether 
this result is due to diminished nitrogen fixation or to increased loss of 
nitrogen cannot be determined, but at any rate it disposes of the 
hypothesis that partial sterilisation is followed by an increase in the 
total nitrogenous matter in the soil. (§ 18, Table 4.) 

§ 3. The two significant changes induced by partial sterilisation 
are thus seen to be (1) an increase in the amount of ammonia, (2) cessa- 
tion of the nitrifying process. 

The accumulation of ammonia which we have shown to take place 
in the treated soils is not simply due to the cessation of nitrification, 
for the amount of ammonia produced is greater than the sum of the 
ammonia and nitrate in the untreated soils after the same period. This 
accumulation may be due either (1) to an increased production of 



116 Partial Sterilisation of Soil 

ammonia in the treated soils, or (2) to the removal by the treatment of 
some agent, other than the nitrifying organisms, which is always con- 
suming ammonia in the untreated soil. The second supposition falls 
to the ground, because when small quantities of ammonium salts are 
added to untreated soils the whole of the added nitrogen is recovered 
as ammonia and nitrate. (§ 2] , Table 8.) 

Hence we conclude that the treatment has induced an increased 
production of ammonia. 

II. The part played by bacteria. 

§ 4. We have confirmed Hiltner and Stormer's discovery that 
bacteria multiply more rapidly and reach far higher numbers in the 
partially sterilised than in the untreated soils. Our untreated soils 
usually contained about 5 to 9 million organisms per gram, a number 
which remained fairly constant. Treatment with toluene effected 
a considerable reduction, but subsequently, when the toluene had gone 
and moisture was added, a period of rapid multiplication set in, and 
the numbers rose to 40 millions or more. The numbers of bacteria 
increase pari passu with ammonia production, and we may therefore 
associate the increased ammonia production with the increased numbers 
of bacteria. (§ 22, Table 9.) 

§ 5. Examining this conclusion in some detail, there is no evidence 
that the species surviving the treatment have received a stimulus 
which makes them more active or that they are the more active sur- 
vivors of a mixed race. The contrary is rather the case ; for instance, 
B. mycoides, and the brown and white streptothrix, were isolated from 
the toluened soil, and all proved less active than the same organisms 
obtained from the untreated soil. (§ 26.) On the other hand we 
obtained considerable evidence that the whole surviving flora is more 
active than the original one in effecting the decomposition of nitro- 
genous organic substances such as peptone, etc., and in hydrolysing 
urea. (§§ 24 and 27, Table 10.) 

Not only is the whole soil with its flora more active, but also the 
flora carried in an aqueous extract of the soil. (§ 25.) The extract is 
prepared at ordinary temperatures by shaking soil with water and 
filtering through cotton-wool ; it contains all organisms that are readily 
detached from the soil and sufficiently small to pass through the filter. 
Such an extract prepared from the partially sterilised soils proved 
more active than extracts of untreated soil in decomposing peptone. 



E. J. Russell and H. B. Hutchinson 117 

§ 6. Examination of gelatine plates prepared by Koch's method 
shows that the flora which establishes itself in the soil after heating is 
altogether different from that originally present, but, on the other hand, 
the flora of the toluened soil did not appear to have markedly altered. 
It is true that certain species were completely suppressed by toluene, 
but their number was only small : indeed out of 27 found in the 
untreated soil only three failed to appear in the toluened soil. Of 
these the most striking is a fluorescent organism, which however did 
not appear to influence the changes one way or the other. (§ 36.) 
Further, of the two streptothrix varieties, the brown predominated in 
the untreated soil and- the white in the toluened, but their difference 
does not appear to be significant. (§ 26.) The curves for ammonia 
production in the heated and toluened soil (Curve 1) are very much 
alike, whilst the bacterial flora is very different: the curves for ammonia 
production in the toluened and untreated soil are fundamentally 
different, whilst the bacterial flora is not. We cannot therefore 
attribute the difference in the rate of ammonia production to a change 
in the type of bacterial flora. 

Our experiments indicate that the increased ammonia production in 
the partially sterilised soil is due to the increased numbers of the 
bacteria. The problem reduces itself to finding out why the bacteria 
can increase so much more rapidly in the partially sterilised, than in 
the untreated soils. 

§ 7. Further evidence that the comparative inertness of the bacteria 
in the untreated soil cannot be caused by any bacterial factor is afforded 
by the following considerations : 

(a) If a filtered soil extract containing bacteria from an untreated 
soil is added to a toluened soil there is an increase in the rate of 
ammonia production, and also in the number of bacteria. 

(b) But if untreated soil is added to toluened soil there is no 
increase in the rates of ammonia production or of bacterial multiplica- 
tion, but, on the contrary, a reduction. These results are set out on 
Curve 3, Table 13, § 36. 

(c) As pointed out above, an extract of toluened soil is more active 
than an extract of untreated soil. 

(d) But when the extract of toluened soil is added to the untreated 
soil there is no increase in ammonia production. 

The conclusion may be drawn that the untreated soil contains a factor, 
not bacterial, limiting the development of bacteria, this factor being put out 
of action by toluening or heating. 



118 Partial Sterilisation of Soil 



III. The nature of the limiting factor. 

§ 8. The limiting factor is not a toxin such as are postulated by 
Whitney and others. 

(1) If it were, it would be sure to affect the nitrification bacteria 
most as they are more sensitive than the ammonia producing groups, 
as seen : 

(a) in their absence from toluened or heated soils, 

(b) in the fact that they cannot be reintroduced into a heated 
soil because the heating has developed some substance toxic to nitrify- 
ing organisms, but not to ammonia producing organisms. (§ 31.) 

In the untreated soil nitrates but never ammonia accumulate, and the 
rate of nitrification is at least as great as the rate of ammonia produc- 
tion. If there is nothing toxic to the nitrifying organisms, a fortiori, it 
is very unlikely there is anything toxic to the ammonia producers. 

(2) Barley seedlings grown in aqueous extracts of untreated and 
toluened soils with or without addition of culture solution showed 
no difference in growth over a period of four weeks. Had any toxin 
been present it should according to Whitney have produced an effect in 
much less time. 

§ 9. The limiting factor is probably biological, since when untreated 
soil is added to toluened soil the reduction in the rate of ammonia pro- 
duction is not at once operative. (Curve 3 (p. 140), § 36.) It is probably 
also a large organism, since it is only the soil and not the filtered extract 
of the untreated soil that is effective in reducing the rate of ammonia 
production in toluened soil. (Cf. § 7a, also §§ 38 and 39.) Search was 
therefore made for large organisms such as infusoria, amoebae, and 
ciliata. None were found in the heated soil, and only small ciliate 
infusoria in the toluened soil. All these organisms are found in the 
untreated soil. Some, e.g. Colpoda cucullus and Amoeba nitrophila, are 
known to devour bacteria, and all must be severe competitors by reason 
of their large size (about 1000 times that of the soil bacteria). We 
conclude then that these large organisms — protozoa, etc. — constitute the 
factor, or one of the factors (see § 42) limiting the bacterial activity, and 
therefore the fertility of our untreated soil. Direct evidence is furnished 
by inoculating toluened soil or soil extract with cultures of large 
organisms, and studying the effect produced. Curve 4 shows the conse- 
quent depression in the rate of ammonia formation. (§ 39, Table 14.) 



E. J. Russell and H. B. Hutchinson 119 

§ 10. We are now in a position to account for all the changes 
brought about by partial sterilisation. 

The micro-organic flora of the ordinary arable soil with which 
we started is very mixed, and includes a wide variety of organisms per- 
forming very different functions. For our purpose they may be divided 
roughly into two classes : saprophytes, which live on and effect the 
decomposition of organic matter and a class comprising 

(a) phagocytes which consume actual living bacteria, 

(b) large organisms inimical in other ways to bacteria. 

The action of the saprophytes tends to increase the fertility of the 
soil, e.g. they produce ammonia, fix nitrogen, and so on. It is true 
that some of them bring about liberation of free nitrogen during the 
decomposition of organic matter, and are to this extent injurious, 
but such action is either much restricted, or is counterbalanced by the 
fixation process, and does not affect our general statement. The 
phagocytes, and similar organisms, on the other hand, must be detri- 
mental to fertility because they limit the number of the organisms 
and therefore the rate of ammonia production. 

Between these two classes of organisms there is an equilibrium 
under natural conditions ; the bacteria cannot multiply indefinitely, 
but are kept in check by the phagocytes ; the phagocytes, on the other 
hand, are kept in check by the limited amount of food, and no doubt 
also by other adverse conditions, such as lack of water 1 . In these cir- 
cumstances bacteria effect only a limited amount of decomposition, 
much less, in fact, than might be expected from the total amount of 
organic matter present. 

When toluene is added, or when the soil is heated to 98°, the 
phagocytes are killed, but the bacterial spores are not. On removing 
the toluene and adding moisture, the spores germinate and the resulting 
organisms multiply with great rapidity, since they are now freed from the 
attacks of their enemies and the competition of other large organisms ; 
they even appear to decompose the dead organisms. There is evidence 
to show that the individual species may be less virulent than the old 
races ; but they more than make up for any deficiency in this direction 
by their enormously increased numbers. The rate of decomposition is 
considerably hastened, and a largely increased amount of ammonia is 
produced. Some of the groups of organisms suffer, such as the 

1 On this view it is easy to explain Rahn's results, which have hitherto remained very 
obscure. He found that drying the soil at ordinary temperature increased its productiveness 
but did not cause what he considered sufficient alteration in the bacterial flora or the food 
supply (i.e. the immediate food supply), Centr. filr Bakteriologie, 1908, n. Bd. 20, S. 38. 



120 Partial Sterilisation of Soil 

nitrogen fixers (§ 29), whilst the nitrifying organisms are absolutely 
exterminated. 

It might be thought that the removal of nitrifying organisms would 
seriously interfere with the growth of plants, but, as a matter of fact, 
it seems to have but little effect ; plants readily take up the decom- 
position products — ammonia, etc. Nitrification is shown to be economi- 
cal, but not essential. (§ 44.) The excess of nitrogenous plant food 
in the partially sterilised soil soon becomes so great that it causes 
a correspondingly vigorous plant growth. 

§11. Partial sterilisation has been found to increase fertility on 
many types of soil and always by increasing the supply of nitrogenous 
plant food. There is reason to suppose therefore that the large destruc- 
tive and competing organisms will be found of common occurrence on 
ordinary soils, checking the beneficent bacteria and limiting fertility. 
An important practical problem arises : is it possible to suppress them 
in ordinary field soils by any economical and practical process ? This 
problem is under investigation. It is unnecessary at this stage to 
enlarge on the importance both from the practical and scientific point 
of view of these large organisms as factors in soil fertility. A fuller 
study of them will no doubt throw much light on many soil problems, 
at present obscure. We are now engaged in further investigations 
of these organisms. 

§ 12. Our results may be summarised as follows : 

(1) The increased productiveness of partially sterilised soils is 
due to an increase in the amount of ammonia present. 

(2) The excess of ammonia is the result of increased decomposition 
of soil substances by bacteria. 

(3) Hiltner and Stormer's discovery that the bacteria increase 
rapidly after partial sterilisation, and finally become much more 
numerous than in the original, untreated soil, is confirmed. The increase 
in number proceeds pari passu with the increase in ammonia. 

(4) The new bacterial flora arising after partial sterilisation is 
a more potent decomposing agent than the original flora, but the in- 
dividual species have not become more, but apparently less potent. 
The increased decomposing power of the new flora is associated with its 
numerical superiority over the old flora. 

(5) The rates of decomposition and of bacterial increase in the 
toluened soil were found to be adversely affected by the addition of the 
original untreated soil. The original soil therefore contains some factor 
which limits bacterial action. 

(6) Chemical hypothesis having been found unsatisfactory the 



E. J. Russell and H. B. Hutchinson 121 

factor is shown to be biological. Large organisms (protozoa) were 
found in the untreated, but not in the partially sterilised soils, at 
least two of which are known to destroy bacteria. 

(7) These large competing and destructive organisms are killed by 
heat and most of them by toluene, and can then serve as food for 
bacteria. In both these directions the effect of partial sterilisation 
is beneficial. 

(8) As the effect of partial sterilisation in increasing productiveness 
is shown on so many soils, and apparently always in the same way, 
it may be expected that these competing and destructive protozoa 
are of common occurrence and constitute an important factor in soil 
fertility. 

(9) In relation to plant growth partially sterilised soils are peculiar 
in that they supply not nitrate, but other nitrogen compounds such as 
ammonia, to the plant. The nitrifying organisms will develope if they 
get into the toluened soil, but they did not work in our heated soils. 
With this difference in the course of nitrogen nutrition may be cor- 
related the difference in nitrogen content of the plant and in the 
character of growth. 

Part 2. 

Experimental. 

§ 13. Crop results. The soil was taken from the outside strip of 
Barnfield, and had been unmanured for many years. It was brought 
down in quantity to the plant-house, spread on a clean cement floor, 
sieved, and carefully picked over to remove worms. The picking over 
requires great care and is very laborious ; unless it is properly done 
the crop weights in duplicate experiments are likely to be discordant. 
The soil was next weighed into pots, tipped out, and mixed with 10 per 
cent, of sand ; then it was either replaced, heated to 98° C. in a large 
steam oven, or treated with toluene, according as it was to be an 
" untreated," a " heated," or a " toluened " soil. In the latter case 2 c.c. 
of toluene were added for every kilogram of soil, left to act for three 
days, then allowed to evaporate by spreading the soil out in a thin 
layer for a sufficient length of time. Finally all the pots were weighed, 
and water added till 18 per cent, was present, an amount which was 
kept fairly constant throughout the period of plant growth. 

The results obtained with successive crops are given in Table 1. 



122 



Partial Sterilisation of Soil 



Table 1. Crop results obtained on partially sterilised soils. 
1st crop, Bye. 





Weight 

of green 

crop 


Weight of dry 
matter 


Composition of dry 
matter 


Weight of material 
taken from soil 


Soil 


In 

grams 


Relative 
weights 


N 
per 

cent. 


p 2 o 5 

per 
cent. 


K 2 
per 
cent. 


N 
grams 


p 2 o 5 

grams 


K 2 

grams 


Untreated... 
Heated 
Toluened ... 


103-95 
16210 

120-07 


37-14 
59-30 
44-76 


100 
160 
120 


•698 

1-147 

•742 


•59 
•64 
•54 


1-05 
1-28 
101 


•259 
•680 
•332 


0-22 
0-38 
0-24 


0-39 
0-76 
0-45 



2nd crop, Buckwheat. 

Untreated.. 

Heated 

Toluened .. 



44-34 


8-53 


100 


1-179 


1-22 


2-22 


•101 


•10 


56-07 


11-19 


131 


1-270 


1-34 


2-05 


•142 


•12 


40-58 


7-79 


91 


1-166 


1-47 


2-09 


•104 


•11 



•18 
•22 
•16 



The treatment was then repeated, and wheat was grown. The crop 
is still standing, but the differences are of the same order as those 
obtained with rye. 

Plate VIII, Fig. 1 shows the rye and Plate VIII, Fig. 2 the wheat 
crops. On reference to the earlier paper it will be found that these 
results, obtained on an exhausted soil, are very similar to those obtained 
on fertile soils. The crops grown on heated soils are shorter in the straw 
and more compact than the others ; typical internodal lengths in cms. 
are : 



Number of internode, counting 
from soil 


1st 


2nd 


3rd 


4th 


5th 


6th 


Length of ear 




Rye grown on untreated soil . . . 
,, toluened ,, 
,, heated ,, 


5 5 

7 
1 


14 
15 
11 


21-5 

20 
16 


27 

21-5 

23 


38 
29 
28-5 


51 
36 
34 


11 
11 
11 



The chemical changes taking place after partial sterilisation. 

§ 14. Two soils have been used in these experiments : the Barnfield 
soil, alluded to above, and a soil from the outside strip of Little Hoos 
which had until 1902 been in ordinary arable cultivation, and since that 
year has carried the same crop as the rest of the field, but has had 
occasional dressings of artificial manures. The two soils contained : 



E. J. Russell and H. B. Hutchinson 



123 



Barnfield 

Little Hoos Field 



Nitrogen 



•112 °/ 
•178 % 



Loss on ignition 



3-969 % 
4-572 % 



CaCO, 



3-409 °/ 
3-159 °/ 



The chemical investigation was directed mainly to the nitrogen 
compounds, ammonia, nitrates, complex and unstable nitrogen com- 
pounds, and humus. The soil was picked over, passed through a 3 mm. 
sieve, and put up in quantities of 800 grams into large bottles. These 
were then divided into four sets : to each bottle in one set was added 
40 grams of toluene, which was allowed to remain in the soil during 
the whole of the experimental period; a second set also received the 
same amount of toluene, but at the end of three days the soil was 
spread out till the toluene evaporated ; a third set was heated to 98° C. 
for 3 hours, and the fourth was left untreated as control. After these 
various treatments the water content of the soil was brought up to 
15 per cent. Precautions were taken to prevent reinfection, and the 
bottles were kept plugged with cotton-wool to admit air; they were 
stored in a cupboard at about 15° C. At suitable intervals a bottle 
was taken from each set and the determinations above referred to 
were made. 

§ 15. Changes in the amount of ammonia and of nitrate 'present. 
The determination of ammonia in soils is complicated by two factors 
which, however, act in opposite directions : soil has a remarkable power 
of retaining ammonia, and, on the other hand, some of the organic com- 
pounds of the soil are very unstable and readily decompose with forma- 
tion of ammonia. By distilling soil at 12 mm. pressure with water 
containing 2 per cent, of magnesia in suspension we have succeeded 
in reducing these sources of error and obtaining quite satisfactory 
results. 

Although ammonia is the final nitrogenous product of the decom- 
position of organic matter in the soil it is not the final result of the 
bacterial activity in the untreated soil, but is at once changed to nitrate. 
It is therefore necessary to make simultaneous determinations of 
ammonia and of nitrates. 

The results of two of the experiments are given in Table 2. Soil 1 
had two years previously received a complete dressing of artificial manure, 
whilst Soil 2 had been for some years unmanured. 



124 



Partial Sterilisation of Soil 



Table 2. Ammonia and nitrate 'present in partially sterilised soils, 
expressed as parts of nitrogen per million of soil dry at 100° G. 

Soil 1. 15 per cent, of water. 





Nitrogen present as ammonia 


Nitrogen present as nitrate 


Treatment of soil 


At 
begin- 
ning 


After 

7 
days 


After 

15 
days 


After 

31 
days 


After 
150 
days 


At 
begin- 
ning 


After 

15 
days 


After 

31 

days 


After 
150 
days 




2-2 
8-6 
4-2 
4-2 


1-9 
27-0 
27-9 


2-7 
32-6 
34-3 


2-2 
37-0 
34-6 

7-5 


8-3 
83-0 
nil* 


17 
17 
15 
18 


24 
16 
16 


26 
13 
13 
16 


33 


Heated to 98°C 


17 


Toluene evaporated 


73 







* Infected with the nitrifying organism. 





Total nitrogen as nitrate and ammonia 


Treatment of soil 


At 
beginning 


After 
31 days 


After 
150 days 


Gain in 
31 days 


Gain in 
150 days 




19-2 
25-6 
19-2 
22-2 


28-2 
50-0 
47-6 
23-5 


41-3 
100 
73 


9 
24-4 
28*4 

1 


22-1 


Heated to 98°C 


74-4 


Toluene evaporated 

Toluene left in 


53-8 











Soil 2. 1st period : 8 per cent, of water present. 





Nitrogen present 
as ammonia 


Nitrogen present 
as nitrate 


Total nitrogen as 
ammonia and nitrate 


Treatment of soil 


At 
begin- 
ning 


After 

13 
days 


After 

63 

days 


At be- 
ginning 


After 
13 days 


At 
begin- 
ning 


After 

13 
days 


Gain 
in 13 

days 




1-3 

5-0 
4-0 
4-3 


1-8 
6-5 

5-0 

7-2 


1-7 
13-1 
14-5 

20-7 


13 
15 
13 
12 


12 
12 
13 
13 


14-3 

20 
17 
16-3 


13-8 

18-5 

18 

20-2 


nil 


Heated to 98°C 


-1-5 


Toluene left in 


1 
3-9 







2nd period: 17 per cent, of water present. 





Nitrogen present as ammonia 


Nitrogen present 
as nitrate 


Total nitrogen as 
ammonia and nitrate 


Treatment of soil 


At 
begin- 
ning 


After 

2 
days 


After 

4 
days 


After 

9 
days 


After 

23 
days 


After 

54 

days 


At be- 
ginning 


After 

23 

days 


At 
begin- 
ning 


After 

23 
days 


Gain 
in 23 

days 




1-8 
6-5 
5-0 
7-2 


2-0 
7-5 
8-9 
6-7 


2-2 

9-7 

20-0 

8-5 


2-5 
28-1 
22-1 
12-7 


1-7 

43-8 
27-8 
14-5 


trace 
46-9 
34-3 
19-4 


12 
13 
12 
11 


16 
12 
12 
10 


13-8 
19-5 
17-0 
18-2 


17-7 

55-8 
39-8 
24-5 


3-9 


Heated to 98° C. ... 
Toluene evaporated 


36-3 

22-8 
6-3 



E. J. Russell and H. B. Hutchinson 125 

The results for Soil 2 are plotted on Curve 1 (p. 113). 

§ 16. The immediate effect of heating the soil to 95° C. or of 
treating with toluene is to cause a small production of ammonia 
amounting to about 3 to 5 parts per million of soil, and on standing 
there is a further production, the extent of which depends on the 
amount of water present. With 8 per cent, of water the action is slow 
and the curve is linear, but when 17 per cent, is present the curve 
characteristic of bacterial processes is obtained. Action is slow for a few 
days, but by the ninth day it has become very vigorous and remains so 
for a time ; then it slackens considerably. Soil which has been heated 
to 125° C. (at which temperature all organisms are killed) behaves 
altogether differently : after the first production of ammonia due to 
heating there is no subsequent change. It is clear then that the 
continuous formation of ammonia in the partially sterilised soil is due 
to living organisms. 

Where toluene is left in the change is quite different; even with 
17 per cent, of water the curve is nearly linear. The action of micro- 
organisms is in this case excluded, but any enzymes set free could 
continue to bring about decomposition. 

The untreated soil differs from all the others ; there is no accumu- 
lation of ammonia either with 8 or 17 per cent, of water, but there is an 
increase in the amount of nitrate, and the sum of ammonia- and nitrate- 
nitrogen shows a small gain amounting to 9 parts per million in 31 days 
in Soil 1, and 4 parts per million in 23 days in Soil 2. It is known 
that the nitrifying organisms only produce nitrates from ammonia; 
these quantities therefore indicate ammonia that has been formed and 
then nitrified. 

| 17. Unstable nitrogen compounds. When soil is boiled at ordinary 
pressure with water containing magnesia in suspension there is a steady 
and continuous evolution of ammonia arising from the decompositon of 
unstable nitrogen compounds. By working under definite conditions it 
is possible to obtain comparable results ; determinations made simul- 
taneously with those recorded in Table 2 are set out below. The 
immediate effect of toluene and of heat is to increase the unstable 
nitrogen compounds, and is therefore something more than a simple 
liberation of ammonia : there is not however as great a subsequent 
accumulation of the unstable compounds. 

| 18. Total nitrogen. The net change in the amount of nitrogen 
has alone been investigated : it is not at present possible to measure 
the separate processes of fixation and loss. In order to make the 



126 



Partial Sterilisation of Soil 







?H GO 


CO -* 


"? 








go a 


C5 






a 


<j H3 


CM r-< 
















«*h 




rH ip 
CM 4* 


CO 
t- 








<! ^ 


CM i-l 




CO CM 
CO CM 

1 - 1 


o 


CO 


^ CO 

CO p*, 


^H fr- 


** 


=0 


3 


S m s 


CO CM 


O 






o 


<< -3 


CM iH 


1— 1 




5^ 


H 






















^ 




<1 SP-° 


CO CM 


"* 




^ 




(35 t- 


CM 






rQ W 


i-H 


rH 








■2-* >> 


fr- CO 


"? 








IO -tf 






T3 


-<J< T3 


>ra co 


CM 




•ra 


C9 


















,: - 


c3 
•-< 


£l co 
5co >i 


CO 


CM 






ft 

c3 


Vi CM cS 








CO 


<J 13 


Tt< CM 


rH 


t~ CO 


CO 

-hi 


> 
CO 

CO 

a 

CO 

3 








« OS 
CM 


jr! co 
.2 ,-r, ^ 

as 05 cs 


00 iH 
fr- cm 


>b 


e 


<] n3 


CO CM 


rH 




s^ 


o 


















H 


^ 3 bo 


t- O 


c- 




o 
00 

CO 

to 
6 




<j'Ec.3 


O IO 


>o 






-O w 


CM 


1-1 






Sh do 

2-+ >* 


CM Oi 

cb 


CO 




^ 


o 

m 


<t) T3 


t- -^ 


CM 












S 


o 


^B5 >l 


CO CO 


00 










r-l CO 


fr- 




O 


T3 

CO 

"cl 


<1 Tj 


CO -# 


rH 


cp T« 

C5 O 


o 








1—1 
8 


CO 


33 5 

««» J? 


U3 rH 

CO CO 






"o 

CO 


<j) T3 


■* CM 


1-1 




-8 
co 


3 6D 

<ri"S).3 


CO ip 

CO CO 


M 

O 






^ -1 


r-l 


rH 




CO 












rO 




Sh qq 












jg-* >> 


CO CO 


c- 




8 

o 




15 'S 


>o S 


»o 




"3 


^ CO f>i 


co ir- 


OS 




1 


t3 


co 1-1 


■* 


-* 00 














s 


c3 
CO 








1 1 




u 


<J -T3 




CO 




^ 


a 


CO CM 


CO 




co 








CO 




<J 'Sb.9 


C5 00 


i-H 




*!>> 




00 iH 


c- 








JO n 




















« 










. . , 


^_, : 












n ^ 


co 03 : 


o» 










O 'o 

.15 3 


.Si 3 .2 
"2 'a 
a a 


CO 

m 

PQ 






C 


O 

r 


a 


1 ° S 

to ^ 


< 






O cr 


a 




H 






T3 




CO 


10 










a 


.a 








eg 


CO 
























a 








X! 


sa 






















>- 




Q 


C3 





A 






CO 


a 


&s 








a 


■+0 




i-H 








CO 


cS 



« 

§ 



P3 

H 



a t~ 

03 CO 

CO 00 



§^ T^ 



IOOOOOWHO 
CM CM 00 C- -* CM 

CO 00 c^ t- t- fr- 



ee ^ 00 CO -^ CO 
-* lO CO CM 1-1 CO 

1-1 —I O O iH O 



s 

o_ 














*-• s 




CO 


tH 


co "S 




CO 


O 




O 


O 


§ £ 




O 


O 


8 g 




+ 


1 










P 








-a 


ao 


CM 


CM 





c3 O 


•^ 


O 




CO 1— 1 




7-1 


CO . 


g^ 


1—1 


rH 


a 








g 

n3 n 














a co 
53 ft 








O CO ■* CT5 CO O 




OONinaH 




rH i-H rH i-l O i—l 


fc 


i-H rH 


rH rH 


r-i rH 



E. J. Russell and H. B. Hutchinson 



127 



difference as large as possible the soils were kept for some months : the 
results are given in Table 4. 

When Soil 1 was put up it contained 1196 per cent, of nitrogen ; 
during the 15 months the untreated soil has lost "009 per cent. In no 
case does the toluened soil contain more nitrogen than the untreated : 
the assumption that the increased productiveness of partially sterilised 
soils is due to increased nitrogen fixation is therefore wrong. On the 
contrary there appears to be an actually greater loss of nitrogen from 
the toluened soil, and in two cases also from the heated soil than from 
the untreated soil. 

§ 19. Humus. Since humus is a somewhat indefinite group of 
substances the method of determinations must be arbitrary, but by 
working under definite conditions it is possible to get comparable 
results. The soil is washed with dilute hydrochloric acid till the 
washings are free from calcium, then with water, finally it is shaken 
with a 4 per cent, solution of ammonia to dissolve the humus. Deter- 
minations are made of the total organic matter in the extract (humus), 
and of nitrogen left after the ammonia has been boiled off (humic 
nitrogen). 



Table 5. Changes in humus. 

Soil 1, kept 15 months. 





Humus, 
per cent, in soil 


Humic nitrogen, 
per cent, in soil 


Nitrogen in 
humus, per cent. 


Amount originally present 


1-06 
•91 
•93 
•90 


•047 
•049 
•043 
•051 


4-4 


After 15 months, untreated soil... 
Soil heated to 98° 
Toluene evaporated . . . 


5-4 
4-7 
5-6 


The total amount 
Soil 2, kept 10 months. 


of nitrogen is given in Table 4, Soil 1. 




Humus, 
per cent, in soil 


Humic nitrogen, 
per cent, in soil 


Nitrogen in 
humus, per cent. 


Untreated soil 


•90 

■82 
•86 


•046 
•038 
•048 


5-1 


Soil heated to 98° 


4-6 


Toluene evaporated 


5-6 







The experimental error is rather large, and too much stress must 
not be laid upon small differences, but so far as the figures have any 
Journ. of Agric. Sci. in 9 



128 Partial Sterilisation of Soil 

significance they show that the toluened soil loses a little more humus 
than the untreated, but gains a little more humic nitrogen. It is, how- 
ever, quite certain that the toluened soil has not lost any humic 
nitrogen, and the increased ammonia and nitrate recorded in Table 2 
cannot have come from humic nitrogen. 

The heated soil behaves rather differently from the others, but we 
have obtained a good deal of evidence to show that heat decomposes 
humus, and brings about a loss of humic nitrogen. 

The distribution of nitrogen compounds in a typical case is as 
follows : 

Table 6. 





Nitrogen in parts per million of soil 




Ni- 
trate 


Am- 
monia 


Unstable* 
com- 
pounds 


Humic 
com- 
pounds 


Other com- 
pounds (by 
difference) 


Total 


Untreated soil at beginning ... 

,, after 23 days... 
Heated soil at beginning 

,, after 23 days 
Toluened soil at beginning ... 

,, after 23 days ... 


12 
16 
13 

12 
12 
12 


1-8 
1-7 
6-5 

43-8 
5-0 

27-8 


7-1 
4-9 
10-3 
17-8 
14-7 
17-2 


840 
842 
600 

840 
846 


779 

769 

1010 

768 

727 


1640 
1634 
1640 
1640 
1640 
1630 




4 

-1 





37-3 

22-8 


-23 
7-5 
2-5 


2 
6 


-10 


- 6 









-41 













* The " unstable compounds" merge into " other compounds," and the division line is 
purely arbitrary. 

§ 20. Absorption of oxygen. This was investigated by the method 
devised by one of us and described elsewhere 1 . The relative amounts 
absorbed in 10 days by the various soils are given below and show that 
the toluened and heated soils absorb during the first month more than 
does the untreated soil. This result has been previously obtained and 
appears to be quite general. After a time, however, the rate of absorption 
begins to fall off and at the end of 72 days both the toluened and the 
heated soils are absorbing less than the untreated soil. 



1 E. J. Russell, this Journal, 1905, Vol. i. p. 261. 



E. J. Russell and H. B. Hutchinson 

Table 7. Relative absorption of oxygen in 10 days 
untreated soil = 100. 



129 





Untreated soil 


Heated soil 


Toluened soil 


After 28 days 
After 72 days 


100 

87 
63 


100 

145 

25 


206 
102 

51 



§ 21. The increased amount of ammonia in the partially sterilised 
soil is not in itself a sufficient proof of increased ammonia production : 
it might equally arise from a diminished ammonia assimilation if we 
assume the presence of some ammonia or nitrate consuming organism 
in the untreated soil, but not in those partially sterilised. We failed, 
however, to find any evidence of such a process. Periodical deter- 
minations of the ammonia and nitrate in soils to which known quantities 
of ammonium sulphate had been added always accounted for all or more 
than all of the added ammonia. Some of the results obtained are as 
follows : 

Table 8. Effect of adding ammonium sulphate to soil. 





Nitrogen present as 
ammonia 


Nitrogen present as 
nitrate 


Total nitrogen as 

ammonia and 

nitrate 




At 
begin- 
ning 


After 

6 
days 


After 

13 
days 


After 

55 

days 


At 
begin- 
ning 


After 

6 
days 


After 

13 
days 


After 

55 

days 


At 
begin- 
ning 


After 

55 
days 


Differ- 
ence 


Untreated soil . . . 


122-8 


111-0 


91-3 


4-9 


18-5 


18-7 


40-0 


157-2 


141-3 


162-1 


+ 20-8 



These experiments show that (1) the increased productiveness of a 
partially sterilised soil is due to an increase in the amount of ammonia 
present, 

(2) the increased amount of ammonia is the result of bacterial 
action, 

(3) it is not due to a diminished assimilation of ammonia or 
nitrate but to an actual increase in the rate of ammonia production. 

§ 22. The total number of bacteria capable of developing on gelatine 
plates. The first effect of partial sterilisation is to reduce considerably 
the number of these organisms present, but when the soil is subsequently 

9—2 



130 



Partial Sterilisation of Soil 



moistened an enormous increase takes place. This fact was first observed 
by Hiltner and Stormer and has been repeatedly confirmed during the 
course of our investigations ; as an instance the following figures may 
be quoted: 

Table 9. 

Soil 1. Arable soil from Little Hoos Field (§ 14). 





Number of organisms per gram of dry soil 


Ammonia produced 
in 9 days, parts 




At beginning 


After 9 days 


Increase 
during 9 days 


per million of 
dry soil 


Untreated soil 
Toluene evaporated 


6,693,000 

2,608,000 

393 

2,311,000 


9,814,000 
40,620,000 
6,294,100* 
2,617,000 


3,121,000 
38,012,000 
6,294,000* 
300,000 


0-7 
17 1 
3-2* 


Toluene left in 


5-5 



* After 4 days, 9 days' counts lost by plates liquefying. 
Soil 2. Eich garden soil containing -592 per cent, of nitrogen. 



Untreated soil 

Toluene evaporated. 
Heated soil 



At beginning After 10 days 



4,200,000 

1,306,000 

40 



10,600,000 

31,680,000 

7,360,000 



After 38 days 



13,850,000 
38,200,000 
17,600,000 



Plate IX, Figs. 4 and 5 show the photographs of the plates. 

The rapid rate of increase in the partially sterilised soils is not 
maintained indefinitely, but even after some months the toluened soil 
contains more organisms than the untreated soil, the actual excess 
depending on the conditions that have obtained in the meantime. 

A very important relationship is brought out by the above figures. 
It will be noticed that the increase in the number of organisms runs 
parallel with the increase in the amount of ammonia : we may therefore 
infer that the increased ammonia production is associated with the 
increase in the number of bacteria. 

§ 23. The production of ammonia by soil bacteria from nitrogenous 
compounds. For the elucidation of this problem we had recourse in 
the first instance to a method devised by Remy which, after suitable 
modifications, gave very useful results. 

§ 24. The production of ammonia from peptone. In Remy's method 
10 grains of soil are inoculated into 100 c.c. of a culture solution 
containing 1 per cent, of peptone besides nutrient salts, but no ammonia, 



E. J. Russell and H. B. Hutchinson 



131 



and the whole is left for three days in an incubator. The ammonia 
produced is then determined. In most of our experiments made in 
this way inoculation with toluened soil caused about 15 per cent, 
greater production of ammonia than inoculation with untreated soil, 
whilst heated soil only yielded about half as much : the results however 
were not always consistent and it sometimes happened that the toluened 
soil culture gave no more ammonia than the untreated. After numerous 
trials three modifications were finally introduced. 

(1) Instead of stopping the reaction at any arbitrary moment and 
expressing the result as a number we have found it better to make a 
series of determinations at definite intervals and plot the results as 
a curve expressing the rate at which reaction takes place. In this way 
the method becomes more sensitive and gives more useful information. 

(2) The cotton-wool plug was replaced by an acid trap to prevent 
loss of ammonia by volatilisation. 

(3) A stronger inoculation was made, 25 grains of soil being 
introduced into 15 cc. of a 3*3 per cent, solution of peptone. 

Of these the third is purely a matter of convenience ; the general 
type of curve obtained is independent of the strength of inoculation. 
Two experiments made by the modified method are recorded below : 
the figures are plotted on Curve 2 (p. 114). 



Experiment 1. Arable soil as used for determinations given in Table 2. 



Treatment 


Ammonia (expressed as nitrogen) in mgrams produced after 




6 hours 


12 hours 


18 hours 


24 hours 


Untreated soil 


•8 

1-2 


9-8 

10-1 


11-5 


17-3 


26-3 


Soil heated to 98° 


2-7 


Toluened soil 


27-7 







The number of organisms in the culture was determined by nutrient 
agar plate culture : the results, expressed as the number per gram of 
soil present, are: 



Treatment 


At 
beginning 


After 
6 hours 


After 
12 hours 


After 
18 hours 


After 
24 hours 


Untreated soil 

Soil heated to 98°... 
Toluened soil 


28,900,000 

1,000 

2,520,000 


100,500,000 

60,200 

11,700,000 


866,000,000 

814,000 

40,500,000 


933,000,000 

5,280,000 

66,400,000 


1,310,000,000 

9,500,000 

128,800,000 







132 



Partial Sterilisation of Soil 



Repetition of the experiment with another soil and with gelatine 
plates gave similar results. The plates showed sharp differences in 
flora ; fluorescent bacteria predominated on the plates poured from the 
untreated soil cultures but were absent from the others: B. mycoides 
and zopfii were most numerous on the plates poured from the toluened 
soil. It is shown later on that B. mycoides decomposes peptone much 
more rapidly than B. fluorescens. 



Experiment. 2. 


A rich garden soil 














Ammonia in mgrams produced after 


Treatment 


4 hours 


8 hours 


12 hours 


16 hours 


20 hours 


24 hours 


30 hours 


Untreated soil ... 
Heated soil 
Toluened soil ... 


•9 

•5 

2-0 


2-9 

•7 
6-0 


8-8 

2-9 

15 4 


15-0 

5-0 

23 9 


25-5 

6 2 

33-6 


30-1 

8-5 

35-3 


39-9 
15-0 
42-6 



This rate of change is at first slow, then it rapidly increases and 
finally it slackens. The rapid period sets in some time earlier in the 
toluened soil than in the untreated, but is delayed considerably in the 
heated soil probably because of the small number of organisms which 
survive a temperature of 98° C. 

It was found that a little toluene reduced the decomposition rate 
almost to zero, thus affording further proof, if more were needed, of the 
bacterial origin of the change. 

§ 25. It is by no means necessary that soil should be used as the 
inoculating material in these experiments. The filtered liquid from the 
peptone cultures readily decomposes peptone, and in this case also the 
culture obtained from the toluened soil is more potent than that from 
the untreated. 





Ammonia in mgrams produced after 


Filtrate from 


8 hours 


16 hours 


24 hours 


36 hours 


Untreated soil culture 
Toluened soil culture 


0-1 
0-6 


2-7 
1-5 


5-2 
6-2 


8-1 
11-3 



Again, the extracts obtained by shaking some of the soil with water 
and filtering through cotton-wool show the same kind of difference in 



E. J. Russell and H. B. Hutchinson 



133 



the amount of decomposition they bring about when inoculated into 
peptone solution. The bacteria present in the extract of the tokened 
soil are more effective than those in the extract of the untreated soil ; 
the results are : 





Ammonia in mgrams produced after 


Extract from 


12 hours 


18 hours 


32 hours 


Untreated soil ... 
Toluened soil 


1-7 
21 


3-8 
4-2 


10-8 
16-6 



Similar results are obtained when other nitrogenous compounds are 
substituted for peptone. Casein, gelatine and lucerne hay infusion were 
all decomposed more readily by the toluened than by the untreated 
soil. 

It is thus clear that the flora which survives the process of partial 
sterilisation and developes when the conditions again become favourable 
is more effective in producing ammonia from complex nitrogen compounds 
than the original flora of the soil. 

§ 26. This conclusion however only applies to the community of 
organisms considered as a whole. If we isolate any individual species 
of organism we find that the cultures made from the toluened soils are 
actually less potent than cultures of the same organism from untreated 
soils. The amounts (in mgms.) of ammonia produced from peptone 
solution in 76 hours were found to be : 



Organisms from 


B. mycoides 


White 
streptothrix 


Brown 

streptothrix 


Untreated soil 

Heated soil 


10-2 
5-9 


2 5 

2-0 


2-6 
2-2 







It is clear that we must not explain the effects of partial sterilisation 
by assuming that the separate organisms or group of organisms are 
rendered more virulent, or more effective by loss of weaker members, 
or that they are stimulated for any long period by the temporary action 
of the toluene. The contrary indeed happens, and the individual species 
rather suffer by the treatment. An interesting point brought out is 
that the brown and white streptothrix possess very similar decom- 
posing powers. 



134 



Partial Sterilisation of Soil 



§ 27. The production of ammonia from urea. The experiments with 
urea were on the same lines as the preceding and lead to the same 
conclusions. 

Table 10. 





mgrams of ammonia produced 
after 


Bacteria present in millions per 
gram of soil 


Treatment of soil 


8 hours 


16 hrs. 


24 hrs. 


32 hrs. 


hour 


8 hours 


16 hrs. 


24 hrs. 


32 hrs. 


Untreated 


1-7 

2-8 








3-9 

8-3 

2-1 






5-9 

16-5 

4-3 

2-5 

1-4 


6-4 
57-4 
5-3 




2-4 

•78 
2-0 




21 
3-0 
2-1 

•1 



30 
5-9 

2-8 




7-0 

13-0 

2-7 

•9 


1-5 


Toluene evaporated.. 

Toluene left in 

Heated to 98° C, 
Heated to 120° C. ... 


66-0 
2-1 
1-7 




Decomposition has been most rapid in the "toluene evaporated" 
soil but it also, goes on in presence of toluene. It is not due in the 
latter case to catalytic action of the soil since it does not take place 
in the heated soils, but is most probably brought about by an enzyme. 

§ 28. There is a fundamental difference between the decomposition 
of peptone and of urea. Peptone acts as a nutrient, urea does not, but 
is decomposed by a purely fermentative change. The aqueous extract 
of the soils had little or no action on urea solution till peptone was 
added and then decomposition took place : the amount of decomposition 
increased with the amount of peptone added. 

Table 11. Effect of nitrogenous food supply on the rate of urea 
hydrolysis (25 grams untreated soil and 15 c.c. 1 °/ urea solution 
and varying amounts of nitrogen as peptone). 



Nitrogen added as peptone, mgrams 

Mgrams of ammonia, expressed as nitrogen, 
produced after 44 hours 






0-26 


0-52 


1-04 


6-6 


8-0 


13-0 


31-9 



2-08 
59-0 



Under the same conditions toluened soil produced 53'8 mgms. of 
nitrogen as ammonia; a striking proof of its superior decomposing 
powers. 

§ 29. Nitrogen fixation. 5 grams of soil were inoculated into 
50 c.c. of a 2 per cent, mannite solution containing potassium phosphate 
(Beyerinck's solution) and the whole was allowed to stand at 30° in 
an Erlenmeyer flask plugged with cotton-wool for 21 days. As no 
nitrogen compound was supplied those organisms alone could develope 
that take their nitrogen direct from the air. The toluened soil fixed 
less than the untreated, whilst the heated soil fixed practically none. 



E. J. Russell and H. B. Hutchinson 135 

The actual amount of nitrogen fixed per gram of marmite supplied 

was: 

Arable soil Garden soil 

Untreated soil 4-7 6-3 mgrams 

Toluened soil 3-8 6-2 ,, 

Heated soil -5 -2 „ 

These results confirm our previous conclusion that the increased 
productiveness of partially sterilised soils is not due to increased 
nitrogen fixation. 

§ 30. Nitrification. Both heat and toluene destroyed the nitrifying 
organisms ; there was no sign of revival even after a month's incubation 
at 30°. We have already shown (Table 2) that nitrates are not produced 
in partially sterilised soils except as a result of subsequent infection. 

It has of course been known for many years that volatile antiseptics 
put an end to nitrification, but it is usually considered that the nitrifying 
organisms recover after an interval, and even, according to some, work 
at an increased rate. 

We have made a number of experiments on this point, but in no 

instance have we obtained any evidence of recovery when sufficient 

precautions were taken to guard against re-infection. It was a common 

experience that nitrification would be for a long time suspended in 

toluened soils and would then set in with the production of a large 

amount of nitrate, thus: 

At beginning After 6 weeks After 18 weeks 
Parts of nitrogen as nitrate ) . „ 1 „ (64 

per million of dry soil J 1 82 

The large amount of nitrate is of course no evidence that nitrification 
is stimulated, but is simply the result of the increased ammonia 
production, and accidental inoculation with nitrifying organisms. 

§ 31. When the soil has been heated, however, it becomes unfitted 

for the development of the nitrifying organism. Apparently a toxic 

body is produced, which however only acts on the nitrifying organism 

and not on those producing ammonia. In one experiment soil was 

completely sterilised by heating to 130° for 45 minutes and then infected 

by admixture with a trace of ordinary soil; the production of ammonia 

and nitrate was as follows : 

At beginning After 21 days After 50 days 

Nitrogen as nitrate 13-6 14-2 15-6 

Nitrogen as ammonia 5-8 26*9 48-6 

Total (parts per million of dry soil) ... 19 '4 41*1 64-2 

Pickering has already demonstrated the formation of a toxic sub- 
stance by heat, and our results are in complete agreement with his on 
this point. 



136 Partial Sterilisation of Soil 

The toxic substance slowly disappears from the soil and ultimately 
nitrification once more becomes possible (cf. also § 45). 

§ 32. Denitrification. Organisms decomposing or assimilating 
nitrates seem to be little influenced by toluene, but they are adversely 
affected, though not killed, by heat. The nitrate completely dis- 
appeared in 5 days from 50 c.c. of Giltay's culture solution inoculated 
with 5 grams of untreated or toluened soil and maintained at a tempera- 
ture of 30°, but it persisted for 20 or 30 days when inoculated with 
heated soil. 

§ 33. Organisms suppressed by partial sterilisation. Even a cursory 
examination of the soil reveals the fact that the bacterial flora has 
altered. Neither the heated nor the toluened soils possess the character- 
istic soil odour : the heated frequently smells somewhat musty and the 
toluened has a faint but quite distinct odour. The toluened soil often 
shows white spots like mould, which proved to be white streptothrix. 

§ 34. Gelatine plate cultures were made by Koch's method of 
untreated and partially sterilised soils immediately after partial steri- 
lisation, and again on the ninth day after moistening. There had been 
the usual enormous increase in number in the " toluene evaporated " 
and, to a less extent, the heated soil : this is recorded in Table 12. 
The organisms present on the various plates, and the proportions 
their colonies form to the whole assemblage, are given in Table 12. 

In the untreated soil the white streptothrix and 8 — 11 predominate 
at first, followed by brown streptothrix and the two organisms 15 and 
18, then come a number of others : moulds, mycoides, zopfii, fiuorescens, 
13, 17, 18, etc., none of which formed 10 per cent, of the colonies on the 
plate. After the soils have been kept moist for nine days there is a 
slight rearrangement: 8 — 11 now predominate, then follow the brown 
streptothrix, then the white and 13, whilst the other organisms remained 
as before, so far as could be judged. 

The order in the toluened soil is different. White and brown 
streptothrix and 8 to 11 suffer less than the others and predominate 
directly after toluening. Nine days afterwards white streptothrix has 
gone ahead very considerably and is the principal organism present 
whilst the brown streptothrix formed less than 20 per cent, of the 
colonies. After a long period the brown streptothrix was much further 
diminished and the chief organisms were 8 to 11, 7 and white streptothrix. 
The difference in appearance of the plates is very striking ; the colonies 
from the untreated soil look mainly brown, whilst those from the 
toluened soils are mainly white. It is curious that brown streptothrix 



E. J. KUSSELL AND H. B. HUTCHINSON 



137 



predominates over the white in the untreated soil, but not in the 
toluened soil. Where toluene is left in, however, the white streptothrix 
slowly suffers. Only three of the bacteria observed are killed by the 
short action of toluene — B. fluorescens, 17 and 18. 

The effect of heat is much more drastic. Streptothrix, moulds, 
B. mycoides, fluorescens, zopfii and others are killed, leaving as survivors 
only 8 to 11, 13, 12, 16, 7, of which 8 to 11 and 13 much outnumber 
the rest. The flora of the heated soils is thus fairly simple. 

Table 12. Relative proportions of colonies on the gelatine plates. 
(1) Immediately after partial sterilisation*. 





Untreated 


Toluene 


Toluene 


Heated soil 




soil 


evaporated 


left in 




Total number of organisms | 
per gram J 


6,693,000 


2,600,000 


2,311,000 


393 










Percentage of colonies 










Above 30 


White 




White 


8—11 




streptothrix 




streptothrix 


13 




8—11 








20—30 


Brown 

streptothrix 


White 
streptothrix 


8-11 




10—20 


18 
15 


Brown 

streptothrix 

8—11 

7 

13 


14 












Below 10 


Moulds 


Moulds 


Brown 


12 




B. mycoides 


26 


streptothrix 


16 




B. zopfii 


B. mycoides 


13 


7 




B. fluorescens 


B. zopfii 


B. mycoides 






7 


and others 


B. zopfii 






12 




and others 






and others 








Absent 




B. fluorescens 


B. fluorescens 


B. fluorescens 






17 


17 


17 






18 


18 

Moulds 


18 

Moulds 

Brown 

streptothrix 

White 

streptothrix 

B. mycoides 

B. zopfii 

14 

15 

19 

20—26 



* Pending complete identifications some of the organisms are provisionally designated 
by numbers. 



138 Partial Sterilisation of Soil 

After nine days the proportions have somewhat changed: — 





Untreated 
soil 


Toluene 
evaporated 


Toluene 
left in 


Heated soil 


Total number of organisms \ 
per gram J 


9,814,000 


40,620,000 


2,617,000 


6,294,000* 


Percentage of colonies 










Above 30 


8—11 


White 
streptothrix 


8—11 


13 
8—11 


20—30 


Brown 

streptothrix 

White 
streptothrix 








10—20 


13 


8—11 
Brown 

streptothrix 

7 
B. mycoides 


White 
streptothrix 




Below 10 


Moulds 


Brown 


7 




7 


Moulds 


streptothrix 


12 




15 


B. zopfii 


13 


16 




14 


14 


18 






18 


15 








B. mycoides 


16 








B. zopfii 


13 








B.fiuorescens 


18 







* Alter 4 days ; 9 days' count lost by plates liquefying. 

§ 35. The differences shown by the flora of the toluened and heated 
soils are much more marked than between the toluened and untreated 
soils and cannot in any case be correlated with the ammonia production 
curves. Attention has also been directed to the apparent loss of activity 
after toluening of the separate species examined (§ 26). We must 
therefore conclude that the change in type is less significant than the 
enormous increase in numbers of the decomposing organisms. 

§ 36. It does not appear that any bacterial factor causes the 
comparative infertility of the untreated soil. Addition of the untreated 
soil extract to toluened soil caused no depression in the production of 
ammonia, or the number of organisms, but on the contrary a considerable 
increase; the organisms contained in the extract have no inhibiting 
effect, but multiply side by side with those present in the toluened soil. 
The extract was prepared by shaking 20 grams of soil with 100 c.c. of 
water and filtering through cotton-wool: nor did inoculation with 
B. fluorescens, the most striking organism suppressed by toluene, have 
any inhibiting effect. On the other hand addition of 5 per cent, of 
untreated soil, although at first without apparent action, after a time 
stopped the further increase in bacterial numbers and in ammonia. 
The results are set out in Table 13 and Curve 3 (p. 140). 



E. J. Russell and H. B. Hutchinson 

Table 13. Number of organisms per gram of soil. 



139 



Treatment of soil 


After 20 days 


After 38 days 


After 61 days 


Not infected : 

Untreated soil 


6,000,000 
28,000,000 
32,000,000 

61,300,000 
32,000,000 
73,300,000 
33,600,000 


7,500,000 
31,800,000 
31,600,000 

45,200,000 
46,900,000 
46,700,000 
30,400,000 


9,500,000 
60,100,000 
67,000,000 

166,600,000 


Toluene evaporated 


,, and sterilised soil extract 
Toluened and infected with 

soil extract 


5 per cent, untreated soil 


48,000,000 
67,000,000 


B . fluoresceins 


B. 9—11 


104,000,000 







Change in ammonia and nitrate. 



Treatment of soil 



Not infected : 

Untreated soil 

Toluene evaporated 

,, and sterilised soil extract 
Toluened and infected with 

soil extract 

5 per cent, untreated soil 

B. fluoresceins 

B.9—11 



Nitrogen as ammonia 


At be- 


After 


Gain 


ginning 


57 days 


2-3 


4-8 


2-5 


4-8 


30-2 


25-4 


7-6 


33-9 


26-3 


7-1 


44-9 


37-8 


7 3 


2-9 


-4-4 


6-7 


31-5 


24-8 


7-7 


32-6 


24-9 



Gain in 
nitrogen 
as nitrate 




-1-1 
-2-9 

+ 5-9 

+ 24-7 

-5-5 

-6-7 



Total gain 
in nitrogen 
as ammonia 
and nitrate 
during 
57 days 



2-5 
24-3 
23-4 

43-7 
20-3 
19-3 
18-2 



§ 37. There is clearly some factor in the untreated soils which 
limits bacterial activity and which is put out of action by heating or 
by treatment with toluene. Other experiments lead to the same 
conclusion. 

Arguments have been adduced in § 8, and need not be here repeated, 
against the view that the limiting factor is a toxin. 

Ammonia produced from urea (as mgms. of nitrogen). 



Treatment 


16 

hours 


24 
hours 


37 

hours 


50 
hours 


Extract of untreated soil (5c.c.) and toluened soil (25gms.)... 
,, toluened ,, ,, ,, 


2-2 
2-2 


7-4 

8-7 


51-0 
52 4 


59-5 
61-0 



§ 38. The limiting factor is probably biological since it takes time 
to operate when it is re-introduced into a toluened soil (§ 36). It occurs 
to a less extent in the extract : thus when an extract of untreated soil 



140 Partial Sterilisation of Soil 

is poured on to toluened soil, the decomposition of urea is only slightly 
less than when an extract of toluened soil is added. 

However, when we applied a more sensitive test and mixed the 
extracts of untreated soil and of toluened soil in equal proportions we 
found that the limiting factor is also present in the extract. 

Toluened soil + aqueous 
/extract containing bacteria 
]0q_ / from untreated soil 



140 



.2 120 



b 
T3 



100 



80 - 



60 



40 



20 ■• 



..Toluened soil alone 

Toluened soil + 5°/. 
untreated soil 



'Untreated soil 



10 20 30 40 50 60 

Time in days 

Curve 3. Effect of untreated soil, and of aqueous extract containing bacteria from 
untreated soil, on the bacterial activity in the toluened soil (Table 13). 

§ 39. These facts point to large organisms as the limiting factor. 
Examination was made for algae and for protozoa by the following 
methods : 

(1) Algae. A solution containing per litre 2 gms. sodium nitrate, 
0'5 gm. each monopotassium, phosphate, sodium chloride, magnesium and 
calcium sulphates was sterilised and inoculated with 5 gms. soil per 
100 c.c. solution, calcium carbonate was also added. The flasks were kept 
in a warm place exposed to light, and after a few weeks a vigorous algae 
growth had developed from the untreated soil, only very little from 
the toluened, and none from the heated soil. Partial sterilisation has 
therefore removed algae. 



E. J. Russell and H. B. Hutchinson 



141 



(2) Protozoa. Soil was inoculated into a sterilised 2 per cent, 
infusion of hay, or, in other experiments, into a sterilised mixture of 
2 per cent, hay infusion and 1*5 per cent, agar which was then poured 
into Petri dishes. After a time large organisms were picked off from 
the untreated soil cultures, including amoebae and ciliata. Some of 
these were kindly examined by Professor S. J. Hickson and found to be 
mainly Colpoda cucullus. The toluened soil cultures only contained very 
small ciliated infusoria, the heated soil cultures contained none. The 
extract of untreated soil generally contained small protozoa. From 
the fact that Colpoda is a common hay infusion form we may infer 
that it is widely distributed and capable of living and multiplying in 
the soil. Its main food seems to be bacteria, and its action must 
therefore be to keep down the number of bacteria and consequently 
the amount of decomposition they effect. We may therefore conclude 
that organisms of this class constitute a factor limiting bacterial activity 
and fertility in ordinary soil. 

Even if certain protozoa and organisms like the algae have no 
direct effect on bacteria they must be severe competitors in the 
struggle for existence in so far as they are actually living in the soil. 
The effect of this large organism is well shown in the following 
experiment on the rate of decomposition of peptone by the extract 
of toluened soil. Addition of an equal volume of the extract of 
untreated soil reduced the rate of decomposition considerably and 
addition of a mixed culture of the large organisms obtained from 
untreated soil brought it down still more. The sterilised extract of 
untreated soil had no effect. 



Table 14. Effect of large organisms on the rate of decomposition 
of peptone by soil bacteria. 





Ammonia in mgms. produced after 




36 hours 


60 hours 


84 hours 


Extract of toluened soil 


2-1 
2-1 

1-6 

2-0 
1-9 


9-8 
5-4 

6-9 

9-8 
6-6 


20-9 


Extract of toluened soil + large organisms from [ 
untreated soil ) 

Extract of toluened soil + extract of untreated ) 
soil, containing large organisms j 

Extract of toluened soil + above extract steri- ) 
lised } 

Extract of untreated soil alone 


8-5 

13-5 

20-8 
12 







142 Partial Sterilisation of Soil 

The results are plotted on Curve 4. 




Extract of Toluened soil 



Extract of Toluened soil + 

Extraet of untreated soil 

(some large organisms present) 



Extract of Toluened soil + 

culture of large organisms from 

untreated soil 



60 80 

Time in hours 
Curve 4. Effect of large organisms from untreated soil on the rate of decomposition 
of peptone by soil bacteria (Table 14). 

§ 40. Not only does partial sterilisation kill these destructive and 
competing organisms and thus make the conditions more favourable 
for the new bacterial flora, but it probably also increases the food 
supply. We have been able to observe under the microscope a dis- 
solution of the killed protozoa by the bacteria. It is not possible as 
yet to form any estimate of the amount of nitrogen thus supplied 
as food, but it cannot be anything like the amount of ammonia 
ultimately produced in the soil. 

§41. As already remarked, toluene does not kill all the large 
organisms but leaves at least one which in course of time developes. 
It is probable that this organism is concerned in the falling off in 
activity of the bacterial soil after a long period as indicated by the 
second crop (Table 1), the drop in the rate of oxidation (§ 20) and 
the fall in bacterial numbers. 

§ 42. While the evidence must be regarded as fairly complete 
that the removal of large unfavourable organisms is one cause of 
the improvement effected by partial sterilisation, we by no means 
wish to imply that it is the only one. It is quite possible that there 
are other factors involved. We found, for instance, a nitrogenous 
substance in the soil which was very soluble in toluene, the distribution 
of which would no doubt be affected by toluening. Some of the 
catalytic changes brought about by soil, e.g. the decomposition of 



E. J. Russell and H. B. Hutchinson 14:3 

hydrogen peroxide, seemed to be influenced by partial sterilisation- 
Heat certainly causes decomposition and increases the food material 
available. These and other factors are under investigation. 

§ 43. Plant growth in partially sterilised soils. So far as the plant 
is concerned the difference between the partially sterilised and the 
untreated soils may be briefly summed up. In the partially sterilised 
soil organic matter decomposes more rapidly with the production of 
a greater amount of ammonia, but no nitrate. Plants make greater 
growth and contain an increased percentage of nitrogen and of phos- 
phoric acid. 

In the earlier paper on partial sterilisation the question was raised : 
In what form do plants take up their nitrogen from partially sterilised 
soils ? The pot experiments indicate that it cannot be taken up as 
nitrate, but they are not conclusive by reason of the liability to 
re-infection. In order to make the evidence quite clear a number 
of plants were grown in conditions where infection did not take place. 

The soil was filled with all proper precautions into sterilised Woolff's 
bottles with three necks. Through the centre neck the sterilised seed 
was dropped and a plug of cotton-wool inserted ; in each of the others 
was fixed a glass tube, one for the water supply reaching to the bottom 
of the bottle, the other, for the air supply, just dipped inside and was 
plugged with cotton-wool. The soil was weighed, and the nitrate was 
determined ; the quantity of nitrate present in each bottle was therefore 
known. The plants were kept in a special glass house kept as free as 
possible from dust. 

Water was added at regular intervals so that 18 per cent, should 
always be present ; the necessary amount was ascertained by weighing 
the whole apparatus on each occasion. The difficulty of adding water 
without at the same time introducing bacteria was overcome by 
permanently connecting a Pasteur flask filled with sterilised water to 
the Woolff's bottle, and transferring water from the flask to the soil 
in the ordinary way. When the crop was harvested at the conclusion 
of the experiment examination was made for the nitrifying organism 
which, however, was found to be absent. The soils were then again 
partially sterilised and sown with a second crop ; the results are given 
in Table 15, and photographs of typical plants in Plate VIII, Fig. 3. 

In a second series of experiments nitrifying organisms were added. 

Six bottles formed the unit in each experiment. 

§ 44. It is quite clear that the plants have got their nitrogen from 
some source other than nitrates. The percentage of nitrogen in the 
dry matter of the rye is at its lowest (= 2'07 per cent.) in all cases 

Journ. of Agric. Sci. hi 



144 



Partial Sterilisation of Soil 



Table 15. 

Series 1. Crops grown without addition of nitrifying organisms. 





1st crop. Rye 


2nd 


crop. Wheat 




Dry 

matter 

produced, 

grams 


Nitrogen 

in dry 

matter, 

per cent. 


Nitrogen 
taken 

from soil, 
grams 


Dry 

matter 

produced, 

grams 


Nitrogen 

in dry 

matter, 

per cent. 


Nitrogen 
taken 

from, soil, 
grams 


Untreated soil ... 
Toluened soil ... 
Heated soil ... 


•836 

1-022 

•994 


2-07 
2-41 
334 


•0173 
•0246 
•0332 


•204 
•885 
•979 


1-612 
1-096 
1-900 


•0032 
•0097 
•0186 



Series 2. Crops grown with addition of nitrifying organisms. 



Untreated soi) .. 
Toluened soil .. 
Heated soil ..... 



•536 


2-11 


•0113 


•353 


1-797 


1-159 


1-99 


•0231 


1-093 


1115 


1-024 


3-18 


•0326 


1-321 


2-029 



•0063 
•0122 

•0268 



Total nitrogen taken by crop, compared with 


nitrogen originally present as nitrate. 




Nitrogen 
in 1st 
crop, 
grams 


Nitrogen 

in 2nd 

crop, 

grams 


Total in 

1st aEd 

2nd crops 


Nitrogen 

as nitrate 

in soil 


Difference, 
being nitrogen 

assimilated 
otherwise than 

as nitrate 


Toluened soil (Series 1) ... 
Heated soil (Series 1) 


•0246 
•0332 


•0097 
•0186 


•0343 

•0518 


•0081 
•0081 


•0262 
•0437 



where nitrate is being produced in the soil and presumably forms the 
chief nitrogenous food of the plant, i.e. in the untreated soil, and the 
untreated and toluened soils inoculated with the nitrifying organism. 
It is higher where no nitrate is being formed, i.e. in the two heated 
soils and the uninoculated toluened soil. The introduction of the 
nitrifying organism into the heated soil has had little or no effect in 
reducing the percentage of nitrogen in the dry matter of the plants. 
Wheat behaves differently, and requires further investigation. 

Nitrification is therefore not essential to plants, but it may be 
economical. A greater weight of dry matter is formed for each unit 
of nitrogen assimilated as nitrate than as other compounds. 

§ 45. Whilst the first crop was growing the nitrifying organisms 
inoculated into the heated failed to develope, being inhibited, probably, 
by a toxic body (§ 31). But during the time the second crop was 
growing the added organisms developed abundantly, a result suggesting 
that the toxic body slowly disappears from the soil. 



JOURNAL OF AGRICULTURAL SCIENCE. Vol. III. No. 2. 



PLATE VIII 






— 


<n 








M 




'1 


«; 




V 














c 




u 






_^: 


E 


B 


B 








_: 


<N rr\ 



60 
Li 








S~o 












-c-o 






S-a 






_^ m 






o 






3 2 


J 




CQ | 






4) U 




5 




d 
o 


-a 

c 


60 


o 


0) 

3 




_c 


3 


-*■ 


H 








t! 


(N 






ho 




t * 








Li, 




■fi a 








' to "O 




— co 












°*o 




~o 


^" 1) 


a3 




M S 


>> 




o-c 


£ 


' 




d 




V> $ 


n 










ta 


h c 


u 


T 


« E 








(A 




>-, ra 






— u 




a 


to £ 

*c 1 — 1 


— 




a 'o 


bu 




o Jr 


Uh 




o . 

>- _c 

00 y 

10 

S..9 

o a. 



JOURNAL OF AGRICULTURAL SCIENCE. Vol. III. No. 2. 



PLATE IK 





(0 

O 





o g -2 a 



B 2 



~— D 4> 0) V 

" ~ « _E _3 



u 



D I H H 



— tN m tt 



cu 

c 




o 






.2 — 

"■S 2 

a; o 

S ~ « 

Of!) 

-S -3 "3 S 



g _ o X- 
2 2 _ -5 g 



— 
w 


T3 


— 

4) 

C 


~3 
U 

c 


V 


u 


V 


u 


'— 


a 


3 


3 



u 



D X H H 



— N <^ ^r 



Lu 



[Reprinted from the Journal of Agricultural Science, Vol. III. Part II.] 
[All Rights reserved.] 



ESTIMATION OF CALCIUM CARBONATE IN SOILS. 

By F. S. MARR, M.A., B.Sc. 

Carnegie Research Scholar. „ - 

Rothamsted Experiment Station. 

This work was undertaken at the suggestion of Mr A. D. Hall, 
whose attention was drawn to the subject by some abnormal results 
obtained in the estimation of calcium carbonate in certain soils from 
different parts of the world characterised by their high humus content 
and their acid reaction to litmus paper. Boiled with diluted sul- 
phuric acid (1 : 1 H 2 S0 4 ), most of these soils yielded an amount of 
carbon dioxide (estimated by Brown and Escombe's double titration 
method) equivalent to a percentage of 1 — 3 of calcium carbonate in 
the air dried soil: while others yielded still higher amounts. It is 
quite possible that a soil may be acid in reaction and yet contain 
carbonate 1 , but such percentages are quite incompatible with the strong 
acidity present in these cases. It seemed possible that the carbon 
dioxide evolved from such soils when boiled with acid resulted from the 
decomposition of unstable organic matter: and this is the conclusion 
arrived at by the writer. 

The apparatus used in the investigation was that described by 
Amos 2 . 

Two soils which showed specially high percentages of carbonate 
(as calculated from the carbon dioxide evolved) were selected as test 
soils. These will be referred to under their Laboratory numbers, Ohio I, 
and Transvaal III: in addition use was made of many other soils both 
acid and normal. They were used in an air dried condition, and powdered 
till they would pass through a sieve with square holes, passing particles 
less than 04 mm. in diameter. The test for decomposition of organic 
matter with production of carbon dioxide was carried out as follows. 
10 grains of soil were placed in a basin with 50 c.c. of boiled water, and 
15 c.c. of strong hydrochloric acid added. The basin was placed in a 

1 Hall, Miller, and Gimingham, Proc. Roy. Soc. B. 1908, 80, 196. 

2 Journal of Agricultural Science, 1905, i. 322. 



156 Estimation of Calcium Carbonate in Soils 

desiccator over strong caustic soda and a good vacuum obtained by 
means of a Fleuss pump. The whole was allowed to stand for several 
hours in order to ensure the decomposition of the carbonate, after which 
time the contents of the basin were washed into the distilling flask of 
the carbon dioxide apparatus with 50 c.c. of water, and boiled for twenty 
minutes. The absorbing Reiset tower was then detached and the 
carbon dioxide estimated. The distillation was then continued for a 
second period of twenty minutes, and also for a third, with the following 
result. The figures are given in milligrams of carbon dioxide per 
100 grams of soil. 



Soil 


1st 20 niins. 


2nd 20 mins. 


3rd 20 mins. 


Transvaal III 

Ohio I 


422 

316 


224 

171 


211 
136 





The results indicate the continued decomposition of something in 
the soil which yields carbon dioxide but which can hardly be calcium 
or any other earthy carbonate. Even if any carbon dioxide remained 
dissolved in the acid solution standing in the vacuum, it would have 
been removed during the first boiling, so that the carbon dioxide obtained 
in the second and third boilings must have been freshly formed by the 
slow decomposition of the organic matter in the soil. 

An attempt was then made to minimise the decomposition of organic 
matter by substituting ammonium chloride for the acid — 

CaC0 3 + 2NH 4 . CI = CaCL, + (NH 4 ) 2 C0 3 . 

Hartleb and Stutzer 1 used ammonium chloride instead of hydrochloric 
acid, and estimated carbonate as ammonia. This method is open to 
criticism, and was found to be quite unreliable for acid soils, the free 
acid of which combines with the ammonia produced and thus renders 
the results too low. In the case of a New Zealand acid soil less ammonia 
came over in the distillation of ammonium chloride with soil than in 
the blank distillation of the ammonium chloride solution itself. 

There can, however, be no objection to a distillation with ammonium 
chloride if the carbon dioxide arising from the dissociation of the 
ammonium carbonate is estimated, and the carbonate calculated from 
this figure: this was done by the writer. 10 — 20 grams of the fine 
soil were put into the distilling flask along with 75 c.c. of boiled water. 

1 Zeit. angew. C'hem. 1899, xn. 448. 



F. S. Marr 



157 



50 c.c. of a 20 °/o solution of ammonium chloride were introduced by 
means of a 3-way funnel, and the distillation was continued for thirty 
minutes after the contents of the distilling flask reached the boil. The 
same apparatus as before was used, with the addition of an acid trap 
containing dilute sulphuric acid and fitted with a condenser. This trap 
was provided to prevent ammonia from reaching the absorbing Reiset 
tower, as it was found that ammonia interfered with the phenol-phthalein 
titration rendering it slower and less sharp. The results obtained by 
this method (expressed as before in milligrams of carbon dioxide per 
100 grams of air-dried soil) were always lower than those obtained 
with hydrochloric acid. 



Soil 


1st 30 mins. 


2nd 30 mins. 


Transvaal III 
Ohio I 


83 
140 


65 

79 





A series of soils yielded on the average 52 milligrams more carbon 
dioxide per 100 grams of soil by distillation with hydrochloric acid than 
with ammonium chloride. The subsoils agreed very closely, a difference 
of only 12 milligrams carbon dioxide per 100 grams soil being obtained 
on the average. This points to the organic matter, which is com- 
paratively speaking absent in the subsoil, as the source of the extra 
carbon dioxide evolved from the surface soil. 

The next step was to ascertain whether by boiling such soils with 
water alone any evolution of carbon dioxide took place. This was in- 
variably found to be the case. The results, calculated as before, are 
given in the following table. 125 c.c. of water was used and the 
boiling continued for 30 minutes. 



Soil 


1st 30 mins. 


2nd 30 mins. 


Transvaal III 


66 
94 
47 
22 


39 


Ohio I 


New Zealand Virgin Pasture... 
Plot 11 Eothamsted Pasture... 



As it was highly improbable that these soils, all of which showed 
a strong acid reaction to litmus, contained any appreciable amount of 
carbonate, and as they gave off carbon dioxide on boiling with water 



158 Estimation of Calcium Carbonate in Soils 

alone, it seemed impossible in such cases to obtain an accurate estimation 
of the carbonate by any method in which the soil was subjected to the 
decomposing effect of water boiling under atmospheric pressure. 

Extraction with ammonium sulphate in the cold was next tried. 
The soil was shaken for twelve or more hours with a strong solution 
of ammonium sulphate, and allowed to stand till the supernatant liquid 
was quite clear. An aliquot portion was then pipetted off by means of 
a filter pump (to avoid disturbing the fine sediment at the bottom of the 
extraction flask), and the carbon dioxide estimated by boiling in Amos' 
apparatus, a little sulphuric acid being added to prevent ammonia from 
reaching the absorbing Reiset tower. While negative results were got 
for carbonate in the acid soils tested, the normal soils always showed 
carbonate though in quantities below those estimated by direct treatment 
with acid. The carbonate could always be determined with a considerable 
degree of accuracy by the following procedure. First of all, the carbon 
dioxide was estimated by Amos' method. The same amount of soil was 
then boiled with dilute hydrochloric acid for a similar period of time 
under like conditions after standing in a vacuum as described in the 
first experiment to verify the decomposition of organic matter. The 
figure for the carbon dioxide evolved from carbonate was found by* sub- 
tracting the amount of carbon dioxide evolved in the latter estimation 
from the total found in the former. The method is not free from 
objection owing to the difficulty of maintaining the experimental con- 
ditions exactly similar, but can be relied on as giving very satisfactory 
results. In normal alkaline soils containing 1 — 2 °/o carbonate of lime, 
the amount of carbon dioxide evolved on boiling with pure water was 
on the average 44 milligrams of carbon dioxide per 100 grams soil 
which corresponds to 01 °/ carbonate of lime. As the ammonium 
chloride method gave results that were much too low in comparison 
with those obtained in the manner described, it was abandoned as 
unreliable. 

Extraction with water supersaturated with carbon dioxide also failed 
to give satisfactory results. The excess of carbon dioxide was boiled 
off and acid added to decompose the precipitated carbonate, but the 
results obtained were very erratic. 

Finally, a distillation with very dilute acid at reduced pressures 
was tried and adopted as giving results which were very satisfactory 
compared with those obtained by distilling the soil under atmospheric 
pressure. Transvaal III, which, as the ammonium sulphate extraction 
showed, contained no carbonate, yielded when boiled with water alone 



F. S. Marr 



159 



under reduced pressure 7 milligrams of carbon dioxide per 100 gm., an 
amount which scarcely exceeds the unavoidable experimental error, and 
certainly shows that water alone did not decompose any appreciable 
amount of organic matter under these conditions. 20 grams of 
the Transvaal soil were now taken and boiled for 20 minutes at 50° C. 
with 2 c.c. strong hydrochloric acid and 100 c.c. water. 19 milligrams 
of carbon dioxide per 100 grams soil were obtained and on continuing 
the process 11 milligrams. The contents of the distilling flask were 
now boiled for 20 minutes at atmospheric pressure and 158 milligrams 
were now evolved. It will be observed that the strength of the acid is 
an important factor in determining the amount of decomposition, as this 
soil yielded 422 milligrams carbon dioxide when boiled with the stronger 
acid used in the test for the decomposition of organic matter. The 
Sprengel water pump was used to reduce the pressure, and considerable 
care must be exercised during the experiment, especially when allowing 
air to pass through the apparatus on the completion of the decomposition 
of the carbonate. 

The results obtained by this method with eight acid soils tested 
never, with the exception of Ohio I and Transvaal III, rose above 
9 milligrams carbon dioxide per 100 grams soil, while on boiling at 
atmospheric pressure ten times as much was found, and that after 
all carbonate must have been decomposed. 9 milligrams of carbon 
dioxide corresponds to 0*02 °/o calcium carbonate, and whether a soil 
contains this amount or no carbonate at all is a matter of no great 
importance. 

The following table gives a comparison of the results obtained for 
the carbon dioxide in Transvaal III and Ohio I b) the various methods 
tried. 



Soil 


With 
1 : 1 H 2 S0 4 


With dilute 
HClat 

atmospheric 
pressure 


With 
NH 4 . CI 
(distilla- 
tion) 


With 
boiling 
water 


With 

NH 4 . CI 

(extraction) 


With dilute 

HC1 under 

reduced 

pressure 


Transvaal III. 
Ohio I 


1540 

2772 


422 

316 


83 
140 


66 
94 






19 
12 





An attempt was made to isolate from Transvaal III and Ohio I 
a portion of the organic matter which, boiled at atmospheric pressure 
with dilute hydrochloric acid, should give off a much larger percentage 
of carbon dioxide than the soil itself. For this purpose part of the 



160 Estimation of Calcium Carbonate in Soils 

humus was extracted with 4% ammonia after preliminary treatment 
with 1 °/ hydrochloric acid, which was removed before extracting with 
ammonia. Curiously enough, the percentage of carbon dioxide evolved 
from the humus, which was dried in a desiccator over sulphuric acid, 
did not increase, although the same experimental conditions were 
maintained as before. 

Amos' observations on the occlusion of carbon dioxide in soil were 
repeated and confirmed. It was found that occlusion of carbon dioxide 
in air-dried soil does not take place to any appreciable extent. 

I have to thank Dr N. H. J. Miller of this laboratory for his 
continued advice and assistance during the progress of this work. 

Summary and Conclusions. 

Boiling acid at atmospheric pressure decomposes organic matter 
in soil with evolution of carbon dioxide, and thus renders the results 
obtained for carbonate too high. Where there is a fairly large percentage 
of carbonate, the error introduced in this way is of no great importance, 
but in soils containing less than 1 °/o°f calcium carbonate and especially 
in acid soils, the error introduced by thus boiling with acid may be very 
considerable. 

The weaker the acid used the better so long as there is fair excess. 
The writer recommends for acid soils and those containing low percentages 
of carbonate (as can be seen by making a rough preliminary test), 2 c.c. 
of strong hydrochloric acid aud about 100 c.c. of water: 20 grams of soil 
should be used when the amount of carbonate is small. The acid may 
be conveniently added by making up a solution containing 100 c.c. of 
strong hydrochloric acid per litre, and introducing 20 c.c. of this solution 
along with 80 c.c. of water. For most soils, 5 c.c. of strong hydrochloric 
acid to 100 c.c. of water will be found convenient. 

If possible distillation under reduced pressure should be used, as 
under this condition practically no decomposition of organic matter 
takes place, while carbonate is readily decomposed: the distillation 
should be continued for twenty minutes at a temperature of about 
50° C. 



Since the above paper was ready for publication we have learnt of the death of 
the author at Breslau on May 13th. After working for a year in the Eothamsted 
Laboratory Mr Marr proceeded to Breslau to work under Dr Th. Pfeiffer, and there 
the course of a promising worker, who endeared himself to all with whom he came 
in contact, was untimely cut short. A. D. H. 



[Reprinted from the Journal of Agricultural Science, Vol. III. Part II] 
[All Rights reserved.] 



THE AMOUNT OF FREE LIME AND THE COMPOSITION 
OF THE SOLUBLE PHOSPHATES IN BASIC SLAG. 

By C. G. T. MORISON, B.A. (Oxon.). 
Rothamsted Experiment Station. 

Basic Slag owes its value as a source of phosphoric acid to the fact 
that it is essentially basic in its character, and can be used on land 
where an acid manure of the character of superphosphate is not to be 
recommended. 

As no figures were available on the subject it seemed interesting to 
determine how much of the lime which it contains existed in the free 
uncombined condition. It has been stated that in some cases this is as 
much as 20 °/o- 

With a view to this determination four samples of freshly ground 
slag were obtained direct from the makers through the kindness of the 
Lawes Chemical Manure Company. 

An attempt was made to follow the method of Stone and Scheuch 1 
for the estimation of lime in commercial quicklime. The method 
consists in shaking a weighed quantity of the slag with a 10 °/ solution 
of cane sugar, filtering and titrating the lime with standard acid. 
However it was found that in the case of some of the slags this solution 
was darkly coloured and quite impossible to titrate, and contained in 
addition to the lime considerable quantities of iron. Further on 
acidifying the solution there was a considerable evolution of hydrogen 
sulphide. It was found that calcium sulphide dissolves to some extent 
in the sugar solution, 100 c.c. dissolving "0174 gram of calcium. 

This method was then abandoned — as were also others depending 
on the reaction of ammonium and sodium carbonates with the lime 
present. In all of these the reaction was interfered with by the sulphides 
present, and by the fact that some phosphoric acid compound was also 
attacked. 

1 J. Amer. Ch. Soc. 1894, xvi. 721. 

11—2 



162 Composition of Basic Slag 

The principle of the method finally adopted is to shake the slag for 
a considerable time with carbon-dioxide-free distilled water, and titrate 
with standard acid, using phenol-phthalein as an indicator. 

The details of manipulation are as follows. A quantity of slag varying 
from 1 to 2 grams is shaken for 24 hours in an end over end shaker 
with 300 c.c. of water freed from carbon dioxide. The whole is then 
poured into a large Buchner funnel and filtered with pressure. The 
time taken for filtration is very small, so that the amount of hydrate 
changed into carbonate cannot be large. The slag is washed back into 
the flask and the process repeated. In the author's determinations 
the extractions were continued until the amount dissolved fell below 
•0008 gram CaO. 

The method probably gives results that are somewhat too low, owing 
to the conversion of a small amount of the hydrate into carbonate during 
the process of filtration, although this is probably compensated for to 
some extent by the fact that other calcium compounds in the slag are 
also to a small extent attacked, as it seemed impossible by continued 
extraction to obtain a solution which was not slightly alkaline to phenol- 
phthalein. 

The point which the author adopted as the limit was usually reached 
at the third extraction. 

Four determinations of lime in the same slag gave the following 
results : 



5-05 
524 
5-22 
5-99 



per cent, free lime. 



It was considered that the results were close enough to make the 
method a useful one. 

In the four samples of slag considered the percentages of CaO were 
as follows : 



A. 


4-69 


B. 


5-29 


C. 


1-28 


D. 


5-37 



The numbers being rather lower than was expected it was suggested 
that this might be owing to the conversion of some of the oxide into 
carbonate as the result of storage. 

Determinations were made of the carbonate present by the method 



C. G. T. Morison 



1(33 



suggested by A. Amos 1 . A tube containing silver sulphate was inserted 
before the absorption Reiset, to prevent the hydrogen sulphide given off 
interfering with the result. 

The figures for the percentage of calcium carbonate in the four 
slags are: 



A. 


2-08 


B. 


214 


C. 


•72 


D. 


•43 



Thus in the four slags examined, which are believed to be typical 
ones, the percentage of lime present both as carbonate and oxide does 
not exceed 7^°/ . 

Table I. 



SLAG 


1st 


2nd 


3rd 


4th 


5th 


Total 
C0 2 sol. 


Total 
in slag 


C0 2 sol. 
°/ of total 


a. j 

Mean... 


5-349 
5-620 

5-484 


3-380 
3-321 

3-350 


1-405 
1-382 

1-393 


•454 
•532 

•493 


•244 

•244* 

•244 


10-832 
11-099 

10-965 


15-81 


66-99 


B. j 
Mean... 


6-080 
6-324 

6-202 


4-310 

4-282 

4-296 


1-880 
1-860 

1-87 


•559 
•7093 

•634 


•259 
•259* 

•259 


13-088 
13-261 

13-179 


18-61 


63-57 


C. 

Mean... 


7-811 
7-166 
7-120 

7-365 


3-471 
3-966 
3-546 

3-661 


•804 

•725 

1-067 

•855 


•210 

•457 
•300 

•322 


•110 
•153 
•110 

•124 


12-307 
12-487 
12-956 

12-584 


18-62 


65-20 


D . { 

Mean... 


5-750 
5-450 

5-600 


5-350 
5-330 

5-340 


2-630 

3-870 

2-750 


•655 
•490 

•572 


•243 
•216 

•229 


14-628 
14-956 

14-792 


22-30 


65-60 



It seemed to be a point of interest to determine what influence this 
amount of free lime had on the action of the solvents employed for 
determining the soluble phosphoric acid, and whether it was correlated 
in any way with the amount of the latter. A solution of carbon dioxide 
was the first solvent employed, and five consecutive extractions were 
made with this on each slag. 

1 Journal Agric. Science, Vol. i. part 3. 

11—3 



164 



Composition of Basic Slag 



The solution was one as far as possible saturated at atmospheric 
pressure by diluting the solution obtained from a sparklet apparatus to 
double its volume and allowing it to stand in contact with the air for 
some time. 

The determinations of phosphoric acid are given in Table I. The 
irregularities in the figures are doubtless due in great part to the 
difficulty in getting a solution of constant composition. 




It will be seen that the quantity dissolved in no case amounts to 
70 °/o of the total phosphoric acid present, and that the proportion is 
very much the same for all the slags. This fact would lead one to the 
conclusion that the easily soluble constituent, whatever it be, is the 
same in each case. 

In Fig. 1 the above results are shown in a graphic form, the 
percentages of phosphoric acid being set out as ordinates, and the 



C. G. T. Morison 



165 



number of extractions as abscissae. It would seem from these that 
had the extractions been pushed further more phosphoric acid might 
have been dissolved out. The difficulties of determining such small 
amounts of phosphoric acid made it impossible to do so. The curves 
are fairly regular except in the case of B and D, which show some 
disturbance, considerable in the case of D, at the beginning. 




This is more clearly seen if, instead of the actual percentages 
obtained, the logarithms of these numbers are plotted, Fig. 2. The 
effect which is evident in the case of all four, but most marked in the 
case of D and B. is doubtless due to the presence of free lime. If the 
carbon dioxide were reacting with a single body these logarithmic curves 
should be straight. As it is it will be noticed that they become so after 
the first or at the furthest the second extraction, and further that those 



166 



Composition of Basic Slag 



containing the most lime show the greatest and that containing the 
least lime the least deflexion. 

The first action of the carbon dioxide would almost certainly be 
the conversion of the free lime into carbonate. The mechanism of the 
following reactions comprising the conversion of carbonate into bi- 
carbonate and the solutions of the phosphoric acid compounds is quite 
obscure. It would be reasonable however to expect, assuming the 
soluble phosphoric acid compound to be the same for the four slags, 
that the slag containing the least quantity of free lime should show 
the highest percentage of phosphoric acid soluble at the first extraction. 
This is precisely what occurs. 

Thus owing to the small mass of the carbon dioxide entering into 
the reaction, the extent to which the phosphates are attacked is masked 
by the presence of varying quantities of free lime. Hence it follows 
that although the natural solvent in the soil is carbon dioxide, it is 
not possible in the case of basic slag to make use of it as a solvent 
for the determination of the soluble phosphoric acid. 

It would seem probable, from the fact that the logarithmic curves 
approach to straight lines and that they run fairly parallel to each 
other, that the substance attacked is essentially the same in all of the 
slags. The probable composition of this compound will be discussed 
later. 

Further determinations were made of the amount of phosphoric 
acid soluble in a 1 °/ solution of citric acid. 

In this case three extractions were made. The results are given 
in Table II. 

Table II. 
Phosphoric Acid soluble in 1 °/ Citric Acid. Shaken for 24 hours. 



Slag 


1st 
extraction 


2nd 

extraction 


3rd 

extraction 


Total sol. 


Total sol. 
Total 


1st extraction 
Total 


A 
B 
C 
D 


13-84 
16-23 
14-06 
20-88 


1-232 

1-098 

•244 

•676 


•052 
•045 
•030 
•015 


15-114 
17-373 
14-334 
21-571 


•9562 
•9331 
•7695 
•9672 


•8754 
•8723 
•7550 
•9366 



Here in presence of a much larger mass of acid the small amount 
of lime has no longer any effect. Further, in 3 of the 4 slags as 
much as 93 — 97 °/ of the total present is dissolved. It is worthy of 
note that in the case of G and D the percentage of total phosphoric 



C. G. T. Morison 167 

acid, soluble in carbon dioxide solution, is the same, whereas in the case 
of the citric acid solution it is vastly different. This would suggest in 
the case of D the presence of compounds unattacked by the carbon 
dioxide. 

It is a well-known fact that the effect of fine grinding, which 
will increase very much the surface in contact with the solvent, has a 
very large effect on the solubility of basic slag. That this has no effect 
on the very soluble phosphates is evident from the results with carbon 
dioxide, as G and D both show the same percentage of total phosphoric 
acid soluble, although the amount of G passing through a 0'2 mm. sieve 
is 76'60, while of D 9821. The effect of the grinding is however shown 
in the citric acid solution. Thus it would appear that in basic slag there 
are at least two sources of phosphoric acid, one of which is very readily 
soluble in a weak acid like carbon dioxide, and one or more which are 
attacked by citric acid to an extent depending on the amount of surface 
exposed. 

A portion of B was finely ground so that the whole passed through 

a 0*2 mm. sieve and citric acid extractions made as before. 

1st 
1st 

B. Original sample 16 '23 

Finely ground sample... 17 '28 

As regards the more difficultly soluble phosphate, as the solubility 
seems to depend so much on the surface of the slag, probably also the 
time during which the two are in contact is an important factor. 

That this is so is seen in the case of B. Two series of determinations 
being made, one with 1 °/o citric acid shaken for 24 hours as above and 
another for \ hour with 2°/o citric acid as recommended under the 
regulations of the Board of Agriculture, the figures are given below: 

\ hour 1st \ hour 

1st 2nd Total 24 hours 1st 24 hours 

A. 24 hours... 14-020 1-810 15-760 

J hour ... 13-13 1-808 14-938 -985 -941 

B. 24 hours... 16-750 1-060 17-810 

£hour ... 14-460 2440 16-900 -949 -692 

It will be seen that the total dissolved may be regarded as the 
same, but considering the first extraction only there is a considerable 
difference and apparently a difference by no means the same for different 
samples of slag. 

The question as to what is the soluble phosphatic compound in basic 
slag has been regarded as settled for a long time. It has always been 



2nd 


3rd 


Total 


Total 


1-098 
1-022 


•045 
•099 


18-61 
18-61 


•8723 
•9286 



168 Composition of Basic Slag 

believed and taught that the body was a calcium phosphate of the 
composition (CaO) 4 P 2 5 . In 1887, Stead and Ridsdale 1 described some 
large and apparently pure crystals of this composition that they obtained 
from basic slag. 

This and a statement of Hilgenstock's seem to be the ground on 
which this belief has been based in spite of the fact that in Jan. 1895 
Stead published another paper 2 in which the former paper is practically 
contradicted. As this latter work seems very generally to have been over- 
looked it may not be out of place to give at some length the conclusion 
arrived at. 

In the first place the author states, "that of the phosphates contained 
in basic slag the most soluble consists of a chemical union of tetra- 
calcium phosphate and mono-calcium silicate. The more insoluble 
phosphates are in the form of hexagonal needles and fiat plates and 
appear to consist essentially of tetra-calcium phosphate, which however 
varies in solubility in different specimens. Some varieties are as insoluble 
as coprolites and nearly as insoluble as apatite." 

The above appears very much at variance with the usual opinion 
of the solubility of tetra-calcium phosphate. 

What really is of still greater importance is the fact that in the 
large number of slags which Stead examined, "there was an entire 
absence of tetra-basic calcium phosphate crystals and a constant 
recurrence of blue crystals " the composition of which he states to be 
(CaO) 4 P 2 5 , CaO . Si0 2 containing 

Ca 56-578%, Si0 2 10-791 %, P 2 5 29146 %. 

Several attempts were made to obtain some crystalline specimens 
of slag. These however proved difficult to obtain, the makers stating 
that crystals were by no means common, and only occurred in certain 
balls of slag. 

Finally, Messrs Albert were able to send a crystalline sample, 
in which however there was no sign of the presence of crystals 
of tetra-calcium phosphate, those present being apparently the blue 
crystals described by Stead, in a more or less pure condition. A sample 
obtained by the author in Berlin showed the same composition. 

The pure blue crystals being very minute it was not easy to obtain 
sufficient for analysis. The time occupied in picking them out was 

1 Trans. Chem. Soc. 1887, 601. 

2 Proceedings of the Cleveland Institute of Engineers. 



C. G. T. Morison 169 

long, as each was examined under a lens and those showing any adherent 
impurity disregarded. 

It was decided to determine only phosphoric acid, calcium, and 
silica. The result is given below : 

Phosphoric acid 26'30 "j 
Calcium oxide 46"7l V 
Silica 1102 J 

These figures being in the ratio of one molecule of phosphoric acid, 
one of silica, and between four and five of calcium oxide. 

The results of other analyses of the crystals which were not so pure 

as the above are given below : 

I II ill 

CaO 38-90 44-20 37-91 

P o 5 19-45 21-53 

SiO, 10-06 10-94 9-36 

FeO 17-03 

The crystals could not be obtained pure in sufficient quantity to 
make a complete analysis possible. 

The points brought out by the above are two: 1st the large amount 
of iron the crystals contain, 2nd the constant molecular ratio of 1 : 5 
between the calcium and the phosphoric acid. 

These analyses would rather point to a body of the general form 
(MO) 5 M 1 . Si0. 3 . P 2 5 where M is calcium more or less replaced by 
ferrous iron, and M x ferrous iron. 

These crystals are very soluble. They dissolve readily in carbon 
dioxide solution, and of the total phosphoric acid present in this slag 
93*2 °/o is soluble in a solvent containing 1 °/ of citric acid, or one- 
twentieth of the concentration usually employed. 

The percentage composition of such a body and the analytical 

figures obtained from the pure crystals are given below : 

Calculated composition Found in 

(CaO) 5 FeO . P 2 5 Si0 2 Crystals 

CaO 50-54 46-74 

FeO 12-99 not determined 
Si0 2 10-83 11-02 

P 2 O s 25-63 26-30 

It will be seen that the figures are fairly well in agreement. The 
material at the author's disposal was not sufficient to enable him to 
proceed further, therefore he merely wishes to suggest the possibility 
of some such constitution as that given above. 

One fact has, however, in the author's opinion been fully established, 
by Stead's work and confirmed by the present analyses that it is not 



170 Comj^osition of Basic Slag 

tetra-calcium phosphate which supplies the soluble phosphoric acid in 
basic slag, but a body in which the molecular ratio of phosphoric acid 
to lime is 1 : 5. 

Consideration of the amounts of phosphoric acid and lime dissolved 
by carbon dioxide solution affords striking confirmation of this as regards 
the whole mass of slag. 

If the first three extractions are considered it may be assumed that 
all the readily soluble bodies have been attacked as well as all the free 
lime dissolved in the form of bicarbonate. The total lime dissolved 
also was determined. 

Sum of 1st three extractions CaO ^2^5 

SlagD 33-48 °/ 13-69% 

If the 5"56 grams of free lime found by the water extraction method 
be subtracted there remain dissolved 27 '92 °/ CaO compared with the 
phosphoric acid. 

The molecular ratio of these is 

27-92 13-69 

56 : H2" 

•498 : -0964 

5 : 1 

Thus of the total lime present in the slag which was 38'62 °/ , 5*8 
was as oxide or carbonate, 27 - 68 was combined in readily soluble form 
leaving 5*17 combined with the remainder of the phosphoric acid. 

V. F. Kroll 1 in a preliminary note says that the principal constituent 
of basic slag is a compound hitherto unknown, consisting of a silico- 
phosphate of lime and ferrous iron, which would seem to agree with the 
results obtained in the present paper. 

The absence of crystals of tetra-calcium phosphate, which were 
undoubtedly obtained from basic slag by earlier observers, and the low 
percentages of free lime now found to be present in the slag, may be 
correlated with the increased percentage of phosphoric acid in slags 
of modern manufacture, less lime being nowadays employed in the 
dephosphorisation process than formerly. 

In conclusion the author wishes to thank the Lawes Agricultural 
Trust for the use of their Laboratory and to express his great indebtedness 
to Mr A. D. Hall, who suggested this investigation, and whose kind 
advice has been invaluable throughout. 

1 Stahl und Eisen, no. 19, May 6, 1908. 



[Reprinted from the Journal of Agricultural Science, Vol. III. Part II.] 
[All Rights reserved.] 



DIEECT ASSIMILATION OF AMMONIUM SALTS 

BY PLANTS. 

By H. B. HUTCHINSON, Ph.D., and N. H. J. MILLER. 

Rothamsted Experiment Station. 

It has recently been shown 1 that the soil of some of the Rothamsted 
Grass Plots which have received ammonium salts for many years in 
succession has become distinctly acid and that, consequently, nitrifying 
organisms have become greatly reduced in numbers. Nitrification is 
limited to portions of soil directly in contact with the few particles of 
calcium carbonate still remaining in the soil. It is evident therefore 
that more or less of the nitrogen assimilated by the grasses must be in 
a form, or in forms, other than nitrate — probably mainly as ammonium 
salt. In view of these results it seemed desirable to obtain additional 
evidence of direct assimilation of ammonium salts by plants. 

The question possesses a further interest in the case of leguminous 
plants, since whilst non-leguminous crops (whether able to assimilate 
ammonia or not) undoubtedly take up, under normal conditions, most 
of their nitrogen in the form of nitrates, we have no knowledge of the 
form of nitrogen appropriated by leguminous plants from their root 
nodules. 

In 1890, Loew 2 showed that platinum black in presence of alkali 
produces ammonium nitrite from nitrogen and water, and suggested 
that assimilation of free nitrogen is accomplished in a similar manner. 
The examination by one of us, in 1890, of numerous fresh nodules 
showed almost invariably an alkaline reaction, sometimes very marked. 
When this view, assigning an indirect role to the nodule organism — the 
production of suitable physical and chemical conditions for the union 
of nitrogen with the elements of water — was put forward, fixation of 
nitrogen apart from the nodules had not yet been observed. Recently 
Loew and Aso 3 have suggested that ammonium nitrite is the first 

1 Proc. Soy. Soc. 1908, B. 80, 196. 2 Ber. 1890, 23, 1447. 

3 Bull. Coll. Agric. Tokyo, 1908, 7, 567. 
Journ. of Agric. Sci. in 13 



180 Direct Assimilation of Ammonium Salts by Plants 

compound produced, and that the nitrous acid is immediately reduced 
to ammonia. An experiment we made with beans taken from a garden, 
showed the presence of ammonia both in the root and in the nodules. 
A few crams of fresh nodules, and about the same weight of the roots 
from which they were taken, were extracted with 75 per cent, alcohol 
and the extracts distilled under reduced pressure with magnesia. The 
amounts of nitrogen as ammonia were as follows : — 

In Roots N. = 0016 per cent. 

In Nodules N. = 0043 per cent. 

If it should be shown that nodules generally contain more ammonia 
than the roots, and that ammonia is readily assimilated by leguminous 
plants, the results would lend some support to Loew's suggestion. In 
this connexion it may be mentioned that Frank (27) looked for nitrates 
in the nodules of peas grown in soil and failed to find any, whilst the 
roots showed a distinct nitrate reaction both above and below the point 
at which the nodules were attached. In the case of plants grown in 
sand free from nitrogen, no nitrates could be detected in any parts. 
Frank also detected the presence of asparagine in lupin and pea nodules 
as well as in the roots. Assuming the initial process in nitrogen fixation 
to be the production of an ammonium salt, it is probable that some of 
the ammonia would at once pass into the roots. It does not follow, 
however, that all the nitrogen derived from the nodules is taken up in 
the same form, and it seems equally possible that the asparagine found 
in the roots may have been partly produced in the roots themselves and 
partly obtained from the nodules. 

Before describing the experiments on assimilation of ammonium 
salts it will be desirable, as the prevailing ideas on the subject are 
anything but clear, to show in some detail what has been already 
done. As, however, the number of papers on the subject is consider- 
able, attention will be confined chiefly to the more recent experiments 
in which nitrification has been taken into account 1 . 

The first experiments in which precautions were taken to avoid the 
possibility of nitrification were made by Pitsch (21) at Wageningen. In 
these experiments, which were commenced in 1885 and continued every 
year until 1894, various plants were grown in humus sand contained in 
metal pots, holding about 30 kilos. The general method employed was 
first to sterilise the contents of the pots, covered with cotton wool, by 

1 The earlier experiments are summarised in S. W. Johnson's How Crops Feed, New 
York, and references are given at the end of this paper. 



H. B. Hutchinson and N. H. J. Miller 181 

suspending in an oil bath heated at 160 — 180°. The soil was next 
extracted (in the pots) with water to remove nitrates, and again 
sterilised. Nitrogen, in the form of ammonium sulphate and sodium 
nitrate respectively, was added to the soil, sometimes both in larger 
and smaller amounts. Occasionally ammonium phosphate was also 
employed. Each series of experiments generally included pots which 
had been neither sterilised nor extracted, as well as sterilised and 
extracted soils without addition of nitrogen. During growth sterilised 
water was supplied to the soil from below. Some time (not imme- 
diately) after the conclusion of the experiments the soil was examined 
for nitrates and in every case nitric nitrogen was found to be absent. 
The results showed that whilst ammonium salts were directly assimi- 
lated, without previous nitrification, the yields obtained with nitrate 
were generally better, the advantage of nitrate over ammonium salts 
being particularly marked during the early stages of growth. 

In an experiment with Oats in 1890, Pitsch found that all the soils, 
at the conclusion of the experiment, contained ammonia (N. = 0001 5 to 
0*0058 per cent.), and that this nitrogen, added to the nitrogen in the 
plants, amounted to considerably more than was contained in the 
manures. It was found moreover that the nitrate plants contained 
more than twice as much nitrogen as was supplied in manure. So 
that these plants evidently drew on the soil nitrogen, probably, for the 
most part, in the form of ammonia, and partly as soluble humus 1 , 
produced in the process of sterilisation. 

In his last experiments, Pitsch shows that additions of sodium 
chloride to the pots manured with ammonium sulphate considerably 
increased the yield. It would seem to be possible that the relatively 
low yields obtained in most cases with ammonium salts may have been 
in part due to unfavourable conditions as regards the mineral con- 
stituents of the soil. 

The methods employed by Pitsch seem to be as satisfactory as 
possible in experiments on so large a scale. It is evident that the 
soils were not only thoroughly sterilised, but that the condition of 
sterilisation was maintained. But although the results show that the 
different plants grew in absence of nitrates, they fail to show that 
the nitrogen assimilated was exclusively in the form of ammonia. 

In 1887, Frank (22) grew beans and sunflowers in water-cultures 
containing nitrogen as ammonium salt and as nitrate. The solutions 

1 Compare H. W. Wiley, Landw. Versuchs-Stat, 1898, 49, 193. 

13—2 



182 Direct Assimilation of Ammonium Salts by Plants 

were not sterilised, and the only precaution to avoid nitrification was 
to add calcium in the form of chloride instead of as carbonate. The 
solutions were found, however, to be free from nitrates and to contain 
ammonia at the end of the experiment. The beans grew fairly well 
when supplied with an ammonium salt, and the stems were found to 
be free from nitrates. 

Mtintz, in 1889 (24) experimented with beans, kidney beans, maize, 
barley and hemp which were grown in soil which was first extracted 
and then heated at 100°. The seeds were sterilised by dipping for a 
moment into boiling water, and the pots were kept in cases ("veritables 
cages de Tyndall") provided with openings, covered with cloth, to render 
the air passing in free from germs. At the conclusion of the experi- 
ment the soils were found to be free from nitrates. The different plants 
assimilated 49 to 915 m.g. of nitrogen, probably in the form of ammonia. 
There is, however, no proof that nitrification had been entirely absent. 
If the ammonium salts had been only slowly, and perhaps locally, nitrified 
all traces of nitrates might have been removed by the plants. In Pitsch's 
experiments as already mentioned, the soils were left for some time after 
the plants were taken out before being examined for nitrates, so as to 
allow time for further nitrification in the event of nitrifying organisms 
being present. 

Griffiths (25), almost at the same time as Mtintz, grew beans in 
sterilised water-cultures, with ammonium sulphate as source of nitrogen. 
The seeds were sterilised by remaining half-an-hour in copper sulphate 
solution, and the jars containing the solutions were placed under large 
bell-jars the openings of which were closed with cotton wool. The 
plants grew remarkably well for four weeks, and reduced the amount 
of nitrogen in the solution from 005 to 0*027 per cent. ; no nitrate 
could be detected. 

The next experiments, by Breal (28), were made with Poa annua. 
Tufts of the grass growing in soil were dug up, and the roots washed 
until free from soil and then placed in water. New roots were soon 
produced, whilst the original roots left off growing. After cutting off the 
old roots the plants were supplied with dilute solutions of ammonium 
sulphate. It was found that after 24 hours all the ammonia had been 
taken up. In these experiments sterilisation was unnecessary as the 
time was too short for nitrification to occur. 

Kinoshita (29), and, subsequently Suzuki (30), grew seedlings of 
various plants for short periods in solutions of ammonium salts and 
sodium nitrate, in order to compare the amounts of asparagine pro- 



H. B. Hutchinson and N. H. J. Miller 183 

duced. It was found that ammonium salts are rapidly converted into 
asparagine, whilst nitrates tended to accumulate, and, during the short 
time the experiment lasted, generally failed to increase the amount of 
asparagine. The production of asparagine is promoted by the presence 
of sugar, and in absence of sugar, or other suitable material, it was 
found that ammonia may accumulate in the plants and eventually 
cause injury. 

In 1898, Maze (32) grew maize in sterilised water-cultures con- 
taining ammonium sulphate and sodium nitrate respectively ; calcium 
carbonate 0*2 per cent, was added. Two months afterwards the plants 
were taken up, and it was found that the ammonium sulphate solutions 
still contained ammonia, and that no nitrate was produced. The plants 
grew about equally well in the two solutions. In later experiments (33), 
culture solutions were employed containing both forms of nitrogen in 
different proportions. The results showed that when the relations of 
ammonium sulphate to sodium nitrate were 1:2 or 1:4 the whole of 
the ammonia was utilised whilst some nitrate remained in the solutions. 

Kossowitsch (35) experimented with peas in sterilised sand-cultures. 
Calcium carbonate was present in addition to the usual minerals, and 
the ammonium salt was added gradually during growth. The results 
showed that ammonium sulphate and sodium nitrate were equally 
suitable as sources of nitrogen. The solutions and sand to which 
ammonium sulphate had been added were found at the end of the 
experiment to be free from nitrates and nitrifying organisms ; in some 
cases, however, it was discovered that other micro-organisms were 
present, and in some moulds. 

Gerlach and Vogel (37) found that maize plants, grown in sterilised 
soil manured with ammonium sulphate, contained more nitrogen 
(0*418 gram.) than similar plants grown in the same soil without 
nitrogen ; the soils were found to be free from nitrates at the con- 
clusion of the experiment. 

Kriiger (38) made a large number of experiments with various 
plants grown in a sterilised mixture of soil and sand. Sterilisation 
was effected by heating the pots in steam for one hour on 6 days; 
the seeds were sterilised with mercuric chloride. At the conclusion of 
the experiment, the soils were examined and those containing nitrate 
excluded. The conclusion is drawn that ammonium salts and nitrates 
are equally suitable for mustard, oats and barley ; that ammonia is, if 
anything, better than nitrates for potatoes, whilst for mangolds nitrates 
are decidedly better than ammonium salts. 



184 Direct Assimilation of Ammonium Salts by Plants 

The last experiments to be described are those of Ehrenberg (39), 
who grew oats in sterilised soil, and in sterilised sand, employing seeds 
sterilised with mercuric chloride. Nitrogen was added in the forms of 
ammonium sulphate and sodium nitrate in sterilised solutions after the 
sand and soil had been sterilised. Calcium carbonate was present. The 
results of both series were negative as regards ammonium salts, the 
plants failing to grow, and the conclusion is drawn that nitrification is 
essential to the growth of higher plants, at any rate in the case of soils 
of slight absorptive power. When, however, the amounts of ammonium 
salts employed are considered in relation to the amount of water present, 
it will be seen that the injurious effects were probably due to too great 
concentration. The sand (5 kilos, per pot) contained 10 per cent, of 
water, or 500 c.c, and the amount of ammonium sulphate present was 
1-4 gram or 2 - 8 grams per litre. In the soil (3"8 kilos.) the amount of 
water was 20 per cent., or 760 c.c, and this contained l - 8 gram of 
ammonium sulphate per litre. It has been shown by Maze (loc. cit), 
that even 1 per thousand of ammonium sulphate is very injurious 1 , 
whilst in the experiment just described the amounts were nearly twice, 
or nearly three times, as high supposing the salt to be equally dis- 
tributed (which was probably not the case), and a good deal higher, 
locally, if not evenly distributed. It is stated that on turning out the 
pots a distinct odour of ammonia was noticed. 

The results of all the experiments described above may be sum- 
marised as follows. The results of Griffiths and Maze' seem to prove 
conclusively that beans and maize assimilate ammonium salts as readily 
as nitrates. The same may be said of Kossowitsch's experiments with 
peas, for although sterilisation was imperfectly maintained, nitrifying 
organisms were completely excluded. Brdal's results may also be con- 
sidered to establish the utilisation of ammonia (by Poa annua). The 
results obtained by Pitsch, Mtintz, Gerlach and Vogel, and Kriiger 
indicate that the various plants employed are able to grow in absence 
of nitrate — not with absolute certainty as regards Muntz's experi- 
ments — but fail to prove that ammonia was the sole source of nitrogen. 

Experimental. 

Seed Sterilisation. In order to obtain vigorous seedlings free from 

nitrifying and other organisms, whose presence would vitiate the results, 

some preliminary experiments on seed sterilisation were made. The 

1 See also Coupin, Rev: Gen. Bot. 1900, 12, 177, and Suzuki, Bull. Coll. Agric. Tokyo, 

1894—7, 2, 265. 



H. B. Hutchinson and N. H. J. Miller 185 

usual method, that of simply soaking the seeds in mercuric chloride 
solution, was found to be unsatisfactory owing to the persistence with 
which occasional air-bubbles remain on or inside the seed, and thus 
prevent complete sterilisation. A greater amount of success was at- 
tained by subjecting the seeds to a preliminary treatment with ether 
or alcohol and subsequent transference to the disinfectant solution. 
The most satisfactory results, however, were obtained by treating the 
seeds in a warm mercuric chloride solution after the removal of any 
air-bubbles by means of a vacuum pump; for this purpose the 
following apparatus was used. 




Fig. 1. 

A stout-walled glass flask B, bearing a rubber cork with two glass 
tubes, was attached on the one hand to a safety flask A, and on the 
other, by means of a three-way tube, to two glass flasks of about 
1 litre capacity G and D. G was filled with a 025 per cent, solution 
of mercuric chloride, D with distilled water. The whole apparatus was 
then sterilised in the autoclave at 125° C. for half an hour, and after 
being allowed to cool to 40° C, the flask A was attached to a vacuum 
pump. Seeds of approximately equal size were then placed in the 
flask B by means of a funnel — to prevent contact between the seeds 
and the neck of the flask — and mercuric chloride was drawn by means 
of the pump into B from G. The connecting tube was then closed 
with a screw-clip and B was evacuated until the solution began to boil. 
By this means all air-bubbles present on the surface of the seed or 
between the cotyledons and the seed-coat were withdrawn, and on 
releasing the vacuum the disinfectant solution was able to act on all 
portions of the seed. 

Sterilisation was allowed to proceed for 3 — 4 minutes, and after B 
had been inverted and the disinfectant withdrawn by means of the 



186 Direct Assimilation of Ammonium Salts by Plants 

pump, sterilised water was allowed to flow in from D and the seeds 
well washed in 2 — 3 changes of water. They were then transferred 
to Petri dishes and a sterilised 1*25 per cent, solution of agar was 
poured in ; solidification of the medium occurred in a few minutes, 
the plates were inverted and placed in the incubator at 20° C. 

At the end of 3 — 4 days, the majority of the seeds had germinated 
and formed roots 1 — \\ inches in length, and if sterile, remained quite 
free from mould or bacterial growth, and were then transferred to 
sterile wide glass test-tubes containing 10 c.c. distilled water over 
which a small plug of cotton wool had been placed. On this cotton 
wool the seedlings were allowed to grow until the shoot was approxi- 
mately 3 inches long, and if they failed to show any subsequent 
infection, were then carried over to the culture bottles at the end of 
7 — 8 days. 

Culture Bottles. Many forms of apparatus have been suggested for 
the cultivation of plants under sterile conditions ; but the majority 
are either too complicated or do not allow sufficient facilities for the 
exclusion of micro-organisms at all stages of the plant's growth. The 
apparatus used in these experiments has the advantage of being com- 
paratively simple, is compact enough to allow of sterilisation in any 
ordinary autoclave, and may be used either for soil-, sand-, or water- 
cultures. 

For the reception of the plant a three-necked Woulff's bottle A of 
750 — 1500 c.c. capacity was taken, and rubber corks were placed in 
each of the side necks. One of the corks held a straight glass tube 
which had at its upper end a small adapter B filled with cotton wool, 
while the lower end almost touched the bottom of the bottle ; this tube 
served to filter the air used for aerating the bottle from time to time. 
The other cork held a short glass tube bent at right angles which was 
connected to a Pasteur-Hansen flask G, filled with distilled water, in 
order that the culture solution in the Woulff's bottle could be kept to 
the same level throughout the course of the experiment. A few drops 
of concentrated sulphuric acid were placed in the side tube D. In many 
cases the flask G was attached to two or three Woulff's bottles by 
means of three- or four-way glass tubes. The middle neck of the 
culture bottle was firmly plugged with cotton wool and the whole 
apparatus heated in the steam steriliser at 99° for three hours. As 
soon as the sterile seedlings had formed shoots about 1\ — 3 inches in 
length they were taken from the test-tubes with sterilised forceps and 
the roots introduced through the middle neck of the Woulff's bottle 



H. B. Hutchinson and N. H. J. Miller 



187 



until they reached the culture solution ; the shoot was then tightly 
plugged round with non-absorbent cotton wool, in order to keep the 
seedling in position. 




Fig. 2. 

Direct Assimilation of Ammonium Salts by Plants. 

Series I. Wheat grown in Sand. The seeds were sterilised in 
0-25 per cent, solution at 45° C. and sown on agar plates. Germination 
was quite normal and after 3 — 4 days the seedlings were transferred 
to sterilised test-tubes and allowed to grow for a further period of 
6 — 7 days. On May 21st, 1908, they were carried over to 10 Woulff's 
bottles containing the following amounts of sand and nutrient salts. 



Sand 

KC1 

KH 2 P0 4 

MgS0 4 + 7H 2 0-10 

NaCl 0-05 

Fe»CL trace 



1200 grams + 2-4 grams CaS0 4 + 2-4 grams Ca.,(P0 4 ) 2 
0-05 gram \ 
0-10 „ 

- dissolved in 50 c.c. distilled water 



Bottles 1 — 3 and 7 — 9 received in addition 6 grams of CaC0 3 . The 
bottles and the Pasteur-Hansen flasks were sterilised in the autoclave 
at 125° C. for half an hour, and after cooling down a solution of 
ammonium sulphate = 21 "98 mgms. of nitrogen was added to bottles 
1_9 ? and sodium nitrate = 20"74 mgms. to bottle 10. 

At the time of transferring the. young sterile plants bottles 7—9 
were inoculated with a culture of nitrifying organisms, and to all the 
bottles 100 c.c. distilled water was added from the Pasteur-Hansen 
flask. From time to time the bottles were weighed and the losses 
made up by adding more water, and aeration was carried out every 
4 — 5 days. 



188 Direct Assimilation of Ammonium Salts by Plants 

The plants in Nos. 7 — 10 grew quite vigorously and possessed a 
dark green colour; Nos. 1 — 3 were also good, while 4 — 6 were stunted, 
No. 6 especially being very poor and ceasing to grow after 12 — 14 days. 
This is shown in the table by the slight amount of dry matter formed 
and of nitrogen assimilated. 

The average amount of nitrogen in each seed was 071 mgm. 



Table I. Wheat in Sand Cultures. 







CaCO : , 
applied 




Dry 


Nitrogen 


Nitrogen 


No. 


Nitrogen applied 




matter 
in crop 


total 
in crop 


in dry 
matter 










gram 


mg. 


per cent. 


1 


Ammonium sulphate 
= 21-98 mgms. N. 


CaCO., 


Sterile 


0-979 


20-72 


2-116 


2 


do. 


do. 


do. 


0-882 


19-72 


2-236 


3 


do. 


do. 


do. 


0-968 


21-07 


2-177 


4 


do. 





do. 


0-648 


15-96 


2-463 


5 


do. 





do. 


1-019 


18-90 


1-854 


6 


do. 





do. 


0-257 


2-03 


— 


7 


do. 


CaC0 3 


Inoculated with 
nitrifying organisms 


1-325 


21-70 


1-638 


8 


do. 


do. 


do. 


1-028 


22-33 


2-172 


9 


do. 


do. 


do. 


1-680 


21-84 


1-300 


10 


Sodium nitrate 
= 20-74 mgms. N. 




Sterile 


0-973 


18-62 


1-913 



At the close of the experiment portions of the sand in each bottle 
were carried over to flasks containing Omelianski's solution and showed 
the absence of nitrifying organisms in bottles 1 — 6. 

Series II. Wheat grown in Water Culture. This series was carried 
out in order to corroborate the results of the previous experiments. 
The treatment of the seed and seedlings was in every respect similar 
to that of the foregoing series, and the seedlings were transplanted 
when about 7 cm. high. Woulff' s bottles of 850 c.cm. capacity were 
fitted with aeration tubes and Pasteur- Hansen flasks and were filled 
with the following solution : — 



+ 1000 c.c. distilled water 



To Nos. 3 and 4, 5 and 6, 2 grams CaC0 3 was added. After the 
bottles had been sterilised in the autoclaves, 10 c.c. of a sterile solu- 



MgS0 4 + 7H 2 


0-5 gram 


CaS0 4 


0-5 „ 


KH 2 P0 4 


0-5 „ 


NaCl 


0-25 „ 


KC1 


0-25 „ 


Fe 2 Cl 6 


10 c.c. of a l°/ solution 



H. B. Hutchinson and N. H. J. Miller 



189 



tion of ammonium sulphate containing 21 "54 mgms. N. was added, 
Nos. 5 and 6 were inoculated with nitrifying organisms from a liquid 
culture, and the sterile seedlings introduced on July 4th in a slightly 
etiolated condition. 

From the commencement of the experiment growth in Nos. 1 and 2 
was very slow, the root growth especially being very poor. During the 
first 3 — 4 weeks, Nos. 3 and 4 grew fairly rapidly and an abundance of 
roots was formed. These however were not equally distributed through- 
out the culture solution but remained in a very coiled mass near the 
surface of the liquid. This marked toxic effect persisted for 4 — 5 weeks 
and was subsequently followed by an even ramification of the roots in 
all portions of the culture solution. 

On August 6th, the plants in Nos. 1 — 5 appeared healthy, while 
that in No. 6 remained etiolated for 2 — 3 weeks and finally died off. 
A marked distinction could be seen in the colour of the plants, that in 
No. 5 being of a much darker green than the others. From August 15th 
the plant in No. 4 began to grow much more vigorously, and the adoption 
of a darker colour seemed to indicate infection with nitrifying organisms. 
This would seem to be supported by the fact that both the dry matter 
is higher and the percentage of nitrogen lower, than in the other 
ammonium sulphate bottles. 

Table II. Wheat growing in Water Cultures. 



No. 






Dry 
matter 


Nitrogen 
total in 


Nitrogen 
in dry 










crop 


matter 








gram 


mg. 


per cent. 


1 


No CaC0 3 


Sterile 


0-284 


7-42 


1-866 


2 


do. 


do. 


0-239 


6-16 


1-841 


3 


CaC0 3 2 grams 


do. 


0-387 


13-02 


2-403 


4 


do. 


do. (?) 


0-872 


14-28 


1-169 


5 


do. 


Inoculated with nitrifying organisms 


1-208 


17-50 


1-035 



Series III. Peas in Water Cultures. The cultures were made in 
Woulff's bottles holding about 1200 c.c. water in which the following 
amounts of the different salts were dissolved : — 



CaSO, 



0'5 gram 



MgS0 4 + 7H 2 


0-5 


KC1 


0-25 


NaCl 


0-25 


KH 2 P0 4 


0-5 


(NH 4 ) 2 S0 4 


0-389 


or NaN0 3 


0-5 


Fe 2 CL 


trace 



190 Direct Assimilation of Ammonium Salts by Plants 

The solutions were sterilised by heating for an hour at 100° on four 
successive days. Calcium carbonate (2 grams) was sterilised, and added 
to each bottle at the same time that the seedlings were put in. The 
bottles were arranged in sets of three, each set being connected with 
a Pasteur flask filled with sterilised distilled water. One set received 
sodium nitrate, and one ammonium sulphate, and there were two similar 
sets which received 2 grams of dextrose in addition, so that there were 
altogether twelve bottles as follows : — 

o 

I. Nos. 1, 2, 3 Sodium nitrate 
II. ,, 4, 5, 6 „ „ + dextrose 

III. ,, 7, 8, 9 Ammonium sulphate 

IV. „ 10, 11, 12 „ „ + dextrose 

The seedlings were put in on June 1, and the plants taken up on 
July 20, 1908. With the exception of No. 8, which failed at an early 
date, all the plants grew normally and showed no appreciable differences 
under the different conditions. Towards the end of the experiment 
No. 6 suddenly lost its green colour owing to the development of a 
mould which quickly appropriated all the available nitrogen. All the 
other plants remained perfectly healthy to the end. 

On taking up the plants it was found that the solutions of Nos. 3, 
4, 5 and 12 were infected. The remaining ammonium solutions were 
free from nitrites and nitrates as well as from nitrifying organisms. In 
the following table are set out the amounts of dry produce, the nitrogen 
in the produce and in the solutions of Nos. 1, 2, 7, 9, 10 and 11. 

Table III. Peas growing in Water Cultures. 



No. 



Nitrate 



Ammonium sulphate 

Ammonium sulphate 
+ dextrose 



Dry 

matter 



grams 
3-194 
2-406 

3-222 
0-860 

2-241 
1-330 



Nitrogen 

in dry 

produce 



per cent. 
2-764 
3-061 

2-819 
5-306 

3-859 
4-679 



Nitrogen 

in 

plants, 

total 



gram 
0-088 
0-074 

0-091 
0-046 

0-086 
0-062 



Nitrogen 
as NH 3 
in solu- 
tion 



gram 



0-003 
0-032 

0-002 
0-018 



Nitrogen 
as N 2 5 
in solu- 
tion 



gram 
trace 
0-007 









Total 
nitrogen 
in solu- 
tion* 



gram 



0-004 
040 

0-007 
0-022 



* Including any nitrogenous matter in suspension. 

The small amount of growth in No. 9 is due to the failure of the 
original seedling; the new plant was consequently a few days behind 



H. B. Hutchinson and N. H. J. Miller 



191 



the others. The number of pods produced by the plants was — (1) 4, 
(2) 5, (7) 3, (10) 2, and (11) 2. 

Additions of dextrose had no appreciable effect, probably owing to 
the presence in the seedlings of sufficient available non-nitrogenous 
material for the production of asparagine from the small amount of 
ammonium salt employed. 

The results of the three series of experiments show that ammonium 
sulphate is directly assimilated by wheat and peas and that, in the case 
of peas, there was no difference between the plants supplied with 
ammonium salt and those which had sodium nitrate. The wheat 
plants, however, showed a decided preference for nitrogen in the form 
of nitrate. 

Percentage of nitrogen in plants manured respectively with 
Ammonium Salts and Nitrates. 

Reference to Tables I, II, and III, will show that in each case in 
which nitrogen was applied as ammonium salts, the dry matter of the 
plants contained higher percentages of nitrogen than when sodium 
nitrate was employed. Maze (loc. cit.), in his water culture experiments, 
obtained similar indications, the percentages of nitrogen being as 
follows : — 

Source of nitrogen N. in dry matter 

Ammonium salt 3*43 °/ 

Sodium nitrate 3*17 °/ 

Table IV. Percentage of Nitrogen in the Mixed Herbage of the 
Rothamsted Grass Plots. 



Plot 


Manuring 


Nitrogen per cent. 


1856—73 


1901—5 


14 

9 
11 

5 


Mixed Mineral Manure and Sodium Nitrate = 86 lb. N. per acre 
„ ,, Ammonium Salts = 86 ,, ,, 
„ ,. =129 „ 
Ammonium Salts alone = 86 „ ,, 


*1-31 
1-55 

tl-74 
2-16 


1-39 
1-52 
1-66 









1858—73. 



t 1856—1861. 



Pitsch also shows (loc. cit.) that in the great majority of cases the 
ammonia plants contain higher percentages of nitrogen than the nitrate 
plants. Further confirmation is afforded by a comparison of the per- 



192 Direct Assimilation of Ammonium Salts by Plants 

centages of nitrogen in the mixed herbage from the Rothamsted grass 
plots, which receive their nitrogen in the form of ammonium salts and 
as nitrates respectively (see Table IV, p. 191). 

Whilst it cannot be assumed that the whole of the nitrogen of the 
ammonia plots is taken up in the form of ammonia, the results as set 
out in the above table increase the probability that much, at any rate, 
of the nitrogen of the crop of plots 5, 9 and 11 is assimilated in its 
original form. 

An explanation of the high nitrogen percentages seems to be afforded 
by Suzuki's results (loc. cit), which showed that ammonium salts are 
rapidly converted by the plants into asparagine, and so give rise to 
conditions favourable to renewed absorption, whilst nitrates tend to 
accumulate and thus check further diffusion from outside. It would 
seem possible that the highly nitrogenous character of leguminous 
plants may have been acquired as a result of long continued nutrition 
with nitrogen, supplied from the root-nodules in a form which lends 
itself to more rapid production of proteids than is possible when 
practically the whole of the nitrogen is taken up as nitrates, as is the 
case with non-leguminous crops. 



Conclusions. 

Agricultural plants of various kinds can produce normal growth 
when supplied with nitrogen in the form of ammonium salts under 
conditions which exclude the possibility of nitrification. Some plants 
grow equally well with ammonium salts or nitrate as source of nitrogen. 
Other plants, while assimilating ammoniacal nitrogen in the absence of 
nitrates, appear to prefer nitrates. It is less certain whether ammonium 
salts can ever produce better final results than nitrates although we 
have indications that this may be the case. 

Lehmann (17) found that whilst buckwheat failed to grow well with 
ammonium salts, maize did far better with this form of nitrogen than 
with nitrates during the first period of growth. Later on the nitrate 
plants recovered, and the ammonia plants became unhealthy, "ein 
Bild des Jammers." Kellner (19) showed that paddy rice also prefers 
ammonium salts to nitrates to commence with, and that nitrates are 
better than ammonium salts for the later growth. The best results 
of all were obtained when both forms of nitrogen were employed 
together. 



JOURNAL OF AGRICULTURAL SCIENCE. Vol. III. No. 2. 



PLATE XIV 




Wheat plants in water-cultures with ammonium salts. 



H. B. Hutchinson and N. H. J. Miller 193 

Plants which take up nitrogen exclusively in the form of ammonium 
salts generally contain very distinctly higher percentages of nitrogen 
than when supplied with nitrates. The question arises whether the 
high percentages of nitrogen in leguminous plants may be due to the 
nitrogen — or most of it — being assimilated in a form more suited to 
the rapid production of proteids than nitrate. 



REFERENCES. 

1. Bouchardat, A. De l'action des sels ammoniacaux sur les vegetaux. Compt. 

rend. 1843, 16, 322-324. 

2. Ville, G. Quel est le role des nitrates dans l'economie des plantes. Compt. 

rend. 1856, 42, 679-683; 43, 612-616. 

3. Cameron, C. A. On urea as a direct source of nitrogen. Hep. Brit. Assoc. 

1857 ; and Chemistry of Agricidture, Dublin, 1857. 

4. Knop, W. Landw. Versuchs-Stat. 1859, 1, 3, and 1860, 2, 65. 

5. Stohmann, F. Henneberg's Jour. f. Landw. 7, 1 ; and Annalen, 1862, 121, 323. 

6. Hellrieoel, H. Ann. d. Landw. 1863, 7, 53, and 8, 119. 

7. Rautenberg, F. und Kuhn, G. Henneberg's Jour. f. Landw. 9, 107. 

8. Birner, H. und Lucanus, B. Wasserculturversuche mit Hafer. Landw. 

Versuchs-Stat. 1866, 8, 128. 

9. Beyer, A. Einige Beobachtungen bei den diesjahrigen Vegetationsversuchen 

in wassrigen Losungen. Landw. Versuchs-Stat. 1867, 9, 480. 

10. — — Versuche iiber die Bedeutung des Ammoniaks, des Harnstofies und der 

Hippixrsaure als stickstofflieferndes Materiel. Ibid. 1869, 11, 267. 

11. Kuhn, G. Notiz iiber das Ammoniak als pflanzlichen Nahrstoft! Ibid. 1867, 

9, 167-168. 

12. Hampe, W. Ueber die Assimilation von Harnstoff und Ammoniak durch die 

Pflanzen. Ibid. 1867, 9, 49 and 157. 

13. Vegetationsversuche mit Ammoniaksalzen, Harnsaure, Hippursaure 

und Glycocoll als Nahrungsmittel der Pflanzen. Ibid. 1868, 10, 176. 

14. Wagner, P. Vegetationsversuche iiber die Stickstoffernahrung der Pflanzen. 

Inaug. Diss. Gottingen, 1869; and Landw. Versuchs-Stat. 1869, 11, 287. 

15. Schloesing. Sur l'absorption de l'ammoniaque de Pair par les vegetaux. 

Compt. rend. 1874, 78, 1700-1703. 

16. Mayer, A. Ueber die Aufnabme von Ammoniak durch oberirdische Pflanzen- 

theile. Landw. Versuchs-Stat. 1874, 17, 329-397. 

17. Lehmann, J. Ueber die zur Ernahrung der Pflanzen geeigneste Form des 

Stickstoffs. Bied. Centr. 1875, 7, 403-409. 

18. Wein, E. Untersuchungen iiber die Form in welcher der Stickstoff den 

Kulturpflanzen zu reichen ist. Bied. Centr. 1882, 11, 152-154; from Zeits. 
landw. Ver. Baiern. 1881, 299-321. 

19. Kellner, O. und Sawano, J. Agriculturstudien iiber die Reiscultur. Landw. 

Versuchs-Stat. 1884, 30, 18-41. 



194 Direct Assimilation of Ammonium Salts by Plants 

20. Harz, C. O. Beitriige zur Stickstofferniihrung einiger Kulturpflanzen. 

Jahresber. k. Thierarzneischule, Miinchen, 1885, 86, 127-162. 

21. Pitsch, 0. Versuche zur Entsekeiduug der Frage, ob salpetersaure Salze fiir 

die Entwickelung unserer landwirtschaftlichen Kulturgewachse unentbehrlich 
siud oder nicht. Landw. Versuchs-Stat. 1887, 34, 217-258 ; 1893, 42, 1-95 ; 
and [with J. van Haarst] 1896, 46, 357-370. 

22. Frank, A. B. Ueber Ursprung und Schicksal der Salpetersaure in der Pflanze. 

Ber. deut. bot. Ges. 1887, 5, 47-54. 

23. Uutersuehungen iiber die Ernahrung der Pflanze mit Stickstoft' und 

iiber den Kreislauf desselben in der Landwirthschaft. Landw. Jahrb. 1888, 
17, 421. 

24. Muntz, A. Sur le role de l'ammoniaque dans la nutrition des vegetaux 

superieurs. Compt. rend. 1889, 109, 646-648. 

25. Griffiths, A. Direct absorption of ammoniacal salts by plants. C/iem. Nevjs, 

1891, 64, 147. 

26. Pagnoul, A. Sur l'emploi de l'azote com me engrais dans les deux formes 

nitrique et ammoniacal. Ann. Agron. 1891, 17, 274-283. 

27. Frank, A. B. Die Assimilation freien Stickstofts bei den Pflanzen in ihrer 

Abhangigkeit von Species, von Ernahrungsverhaltnissen und von Bodenarten. 
Landw. Jahrb. 1892, 21, 1-44. 

28. Breal, E. Contribution a l'etude de l'ali mentation azotee des vegetaux. Ann. 

Agron. 1893, 19, 274-293. 

29. Kinoshita, Y. On the Assimilation of Nitrogen from Nitrates and Am- 

monium Salts by Phaenogams. Bui. Coll. Agric. Tokyo, 1894-7, 2, 200-202. 

30. Suzuki, U. On the formation of asparagine in plants under different con- 

ditions. Bui. Coll. Agric. Tokyo, 1894-7, 2, 409-457. 

31. Muntz, A. Recherches sur rintcrvention de l'ammoniaque de l'atmosphere 

dans la nutrition vegetale. Ann. Sc. Agron. 1896, II, 2, 161-214. 

32. Maze, P. L'assimilation de l'azote nitrique et de l'azote ammoniacal par les 

vegetaux superieurs. Compt. rend. 1898, 127, 1031-1033. 

33. Recherches sur l'influence de l'azote nitrique et de l'azote ammoniacal 

sur le developpement du Mais. Ann. Inst. Pasteur, 1900, 14, 26-45. 

34. Grosse-Bohle, H. Beitriige zur Frage der Selbstreinigung der Gewasser. 

Inaug. Diss. (Minister) Arnsberg, 1900. [Also published by J. Konig, Zeits. 
Untersuch. Nahrungs- u. Oenussmittel, 1900, 3.] 

35. Kossowitsch, P. Ammoniaksalze als unmittelbare Stickstoffquelle fiir Pflanzen. 

J. exper. Landw. 1901, 2, 635. 

36. Treboux, O. Zur Stickstoffernahrung der grunen Pflanzen. Ber. deut. bot. 

Ges. 1904, 22, 570-572. 

37. Gerlach, M. und Vogel, J. Ammoniakstickstoff als Pflanzennahrstoff. Centr. 

Bakt. Par. 1905, n. 14, 124-138. 

38. Kruger, W. Ueber die Bedeutung der Nitrifikation fiir die Kulturpflanzen. 

Landw. Jahrb. 1905, 34, 761. 

39. Ehrenberg, P. Die Bewegung des Ammoniakstickstoffs in der Natur. Mitt. 

Landw. Inst. kgl. Univ. Breslau, 1907, 4, 47-300. 



[Reprinted from the Journal of Agricultural Science, Vol. III. Part II.] 
[All Eights reserved.} 



THE DEVELOPMENT OF THE GRAIN OF 

WHEAT. 

By W. E. BRENCHLEY, B.Sc. 

AND 

A. D. HALL, M.A. F.R.S., 

Rothamsted Experiment Station. 

It is well understood that the grain of wheat is built up out of the 
materials which have previously been elaborated by the plant from the 
crude nutriment drawn from the air and the soil and then stored in 
the stem, roots and leaves until the formation of the seed begins. 
Various observers 1 have followed out the stages in the growth of the 
plant and have determined the periods at which the plant ceases to 
draw nutriment from the soil or the air ; from their investigations it 
would appear that during the latter part of the life of the wheat plant 
the manufacture of fresh material has almost ceased and that the chief 
process going forward is the migration of accumulated material from the 
stem and leaves to the grain. 

For various practical reasons it is important to study this migration 
process in some detail and ascertain the progressive changes in the com- 
position of the grain. For instance, it is very generally supposed that 
if wheat is cut in an unripe condition when the berry is still a little 
green, the grain will yield ' stronger ' flour, i.e. flour capable of yielding 
a larger and better shaped loaf. Again, since the ' strong ' wheats of 
commerce are in the main spring-sown wheats grown in climates which 
become increasingly hot and dry as the season advances, it has been 
supposed that a rapid growth and an accelerated ripening are factors in 
the production of strong wheat. If the first or the last of these 
suppositions are true there remains the further practical question of 

1 J. Pierre, Mem. Soc. Linneenne de Normandie, xv. 1869, 1, 220 ; Deherain, Ann. 
Agron. vm. 1882, 23, xx. 1894, 561 ; J. Adorjan, J. fur Landw. 1902, 50, 193. 

Journ. of Agric. Sci. in 14 



1 96 The Development of the Grain of Wheat 

how far the weight of the produce is affected if the crop is cut 
while still unripe or after it had experienced a premature and forced 
ripening. 

The scientific conception which lay behind these opinions proceeded 
from the observation that grain contained a higher percentage of nitrogen 
when immature than when ripe, whereupon it was concluded that the 
migration of the nitrogenous materials took place first, and that during 
the later stages of the development little besides starch was filled into 
the grain. Thus grain cut unripe would contain more of the nitrogenous 
compounds making up the gluten, which is the chief factor in deter- 
mining the strength of flour. Furthermore, if grain is rapidly grown 
and prematurely ripened time would not be given for the complete 
migration of the starch, and the grain would remain stronger because 
the protein has been less diluted by starch. 

It has also been supposed that as the nitrogenous compounds of the 
grain must enter it in a soluble non-protein form, Avhich gradually 
becomes converted into protein as the ripening process proceeds, 
another reason for the ' strength ' of certain foreign wheats could be 
found in the thoroughness with which the conversion into protein 
had taken place, through the heat of the climates where they were 
grown. 

Such are, or were, the opinions on the ripening of wheat generally 
accepted ; their supposed basis in fact did not however prove trust- 
worthy on experiment. For example, in the experiments of the Home 
Grown Wheat Committee 1 wheat cut in a green state did not yield any 
stronger flour than the same wheat allowed to become dead ripe ; nor did 
variations in the date of sowing from October until March affect the 
strength of the resulting wheat. Moreover, from the numerous trials 
made by that Committee of the strength of foreign wheats grown in 
England and, in one case, of an English wheat grown in Hungary, it 
became evident that the effect of climate in determining the strength 
of wheat has been exaggerated. Strength turns out to be in the main a 
characteristic of the variety, besides which climate, soil and manuring, are 
only minor factors in the result. In consequence of this conflict of opinion 
it was decided to make a re-examination in detail of the progressive 
changes which could be observed in the composition and nature of the 
wheat grain. Not only was the migration of the materials studied by 
analysis but the changes in the intimate structure of the grain and of 

1 Humphries and Biffen, J. Agri. Sci. 1907, n. 1. 



W. E. Brbnchley and A. D. Hall 197 

its constituent cells were followed microscopically. An account of this 
latter part of the work has already been published by one of us 1 ; it will 
be sufficient here to say that no connexion could be traced between 
the progressive changes in the nature of the contents of the cells of 
the endosperm or their final structure, and the strength of the flour 
resulting from the grain. 

The following paper deals with the chemical side of the work. 

Method. In tracing the progressive changes in the migration of the 
materials and the filling up of the wheat grain it is necessary to 
ascertain the total yield on a unit area at a series of dates throughout 
the process, because the same plants cannot both be analysed and also 
allowed to grow on for analysis at a later date. This necessity at once 
introduces a large experimental error : even if comparatively large plots of 
i^th acre, could be harvested at successive dates, the experimental error 
in the yield on each occasion would be not less than 10 per cent., and it 
is increased when the plots are reduced to the very much smaller sizes 
which alone are manageable in work of this kind. Errors of this 
kind vitiated the conclusions reached in certain earlier trials not here 
reported ; in one year particular drills in a wheat field were selected as 
uniform to the eye, and on each date a fixed number of yards of corn 
were cut along these drills ; in another year a hundred good ears were 
selected on each date. The results in both cases led to certain con- 
clusions, but the experimental error was evidently too large, so the results 
have been discarded, though they agree with the data obtained by the 
more accurate methods followed in 1907 and 1908. 

Certain plots of wheat were selected to provide material, and on a 
given day when the wheat was coming into flower all available assistants 
proceeded to mark by means of ties of red wool about 3000 heads of 
wheat which were in just the same stage of development, as shown by 
the fact that they had protruded one or more anthers from the middle 
florets of the ear. Only central stems were marked, never secondary 
tillered shoots ; thus the work began with material as nearly as possible 
uniform and at the same stage of development, From among these 
selected shoots cuttings were made at three-day intervals ; the material 
was brought down to the laboratory and as rapidly as possible the grain 
was picked from the heads. Several lots of 1000 grains were then 
counted out, weighed, dried and weighed again. The bulk of the grain 
was also dried for analysis. Finally all the analyses were calculated 
on the basis of the material contained in 1000 grains, this being a 
1 W. E. Brenchley, Ann. of Botany, Vol. 23, 1909, 117. 

14—2 



198 The Development of the Grain of Wheat 

unit which will suffer the minimum of variation during the whole 
period. 

In the field there will always be a good deal of variation of de- 
velopment between the central and the secondary shoots, hence the 
general produce in the field will not show the progressive changes quite 
so sharply as the experimental material. 

In 1907 one of the wheats was selected from Plot 3 on the Broadbalk 
Field at Rothamsted, which had grown wheat without manure since 
1843 ; the variety was Square Head's Master, a typical heavy-yielding 
weak English wheat. Though the crop on this plot is small, the grain 
is quite normal. Material was also taken from Plot 10 on the same 
field, which receives only nitrogen in the form of ammonium salts every 
year. The grain from this plot shows several peculiarities — it possesses 
a high nitrogen content and looks strong, but when a baking test of 
the flour is made proves to be excessively weak, though after storage for 
some months it gains some strength, without however reaching the 
normal degree for that variety. The third example was taken from 
the neighbouring Little Hoos Field and consisted of spring-sown Red 
Fife, a strong wheat of very different character from Square Head's 
Master. In 1908 only one wheat was selected, this was Square Head's 
Master grown on one of the margins of the Broadbalk Field, which had 
been down in grass some few years before and had also grown potatoes 
with farmyard manure, so that it may be taken to represent wheat 
grown under ordinary conditions of farming. 

The actual data obtained are given in the tables in the Appendix : 
for purposes of discussion they have been thrown into curves, which it 
will be convenient to consider seriatim for each property determined. 
The Red Fife was a few days later both in flowering and cutting than 
the Square Head's Master, but as the march of development was quite 
parallel for the two varieties, the curves which follow have been drawn 
for corresponding periods after flowering instead of for the actual dates 
of sampling. 

The weather conditions prevailing during the two seasons 1907 and 
1908 were in marked contrast ; in 1907 the summer was generally 
overcast and cloudy, with low temperatures and frequent rains ; in 
1908 the early part of the summer was hot, and though there was rain 
in July, August was a fine hot month up to the completion of the 
harvest. 

The following table indicates how different was the weather in the 
two years : 



W. E. Brenchley and A. D. Hall 



199 





Rainfall 


Sunshine 


Temperature 


Maximum 


Minimum 


1907 


1908 


1907 


1908 


1907 


1908 


1907 


1908 


May ,, ., 

June 

July 

August ... 


2-396 
2-609 
2-209 
1-802 


1-886 

1-675 

2-434 

*0-160 


164-5 
160-1 
170-6 
174-5 


198-5 
250-8 
205-1 
*86-7 


59-5 
62-5 
65-1 
66-6 


63-2 

67-9 

69-4 

*69-2 


42-9 
48-4 
48-9 
50-2 


46-2 

48-4 

51-6 

*50-l 



* Up to Aug. 12th, the date of cutting. 

Specific Gravity. In 1907 the specific gravity was determined imme- 
diately the grain had been extracted, by means of a form of volume- 
nometer. The curves obtained in 1907 are set out in Fig. 1: they show 



1-30 
1-25 
1-20 
1-15 








































































,' 


/ 


c;7'"- 


''/ 














S.-* 


.1 


"'? 




/ 






-- 


--«_. 






/. 


/' 


— ' 


^-^ 


/'' 












V10 
1-05 







,- 


«>r~^ 




"~"^ 






/ 













































Plot 3 
R.F. 
Plot 10 





Fig. 1. 



3 6 9 12 15 18 21 24 27 30 33 36 39 days 

Curves showing the specific gravity of the wheat grain at successive 
periods in 1907. 



that though the experimental error is comparatively large there is 
evidently a slight falling off in specific gravity for the first four or five 
periods of three days, after which there is a continual rise up to and 
after the date of cutting. By combining these results with the deter- 
minations of water in the grain at each date it is possible to calculate 
the specific gravity of the dry matter contained in the grain, the mean 
curve of which for all three varieties is given in Fig. 2. From this it is 
evident that the specific gravity of the dry matter falls for about twelve 
days from the beginning of the trials, i.e. until the 22nd day from 
flowering has been reached, after which it remains constant. 



LC 



200 The Development of the Grain of Wheat 

Weight of Grain, Water Content, &c. Fig. 3 shows the green and 
dry weights respectively for each sample. The green weight rises 



'20 

re 

16 


X 


























^ 


^ 


















































14 

12 
























































8 
6 
•4 








































































































































3 6 9 12 15 18 21 ,24 27 30 33 38 39 days 

Fig. 2. Mean curve of specific gravity of dry matter contained in the grain of all 

three plots in 1907. 




12 15 18 21 24 27 30 33 36 39 days 



Fig. 3. Green and dry weights of 1000 grains, 1907 (3 plots) and 1908 (1 plot). 
Upper set of curves represent green weight, lower set dry weight. 






W. E. Brenchley and A. D. Hall 



201 



steadily until about six days before cutting, after whicb it falls off: the 
dry weight rises steadily, though there is little increase in the last six 
days. The riper Square Head's Master even shows a slight but per- 
ceptible decrease in the weight of 1000 grains in the last three days. 
This is probably real and due to the continuance of respiration after 
migration had ceased, though the loss is so small that it falls within the 
limits of experimental error. It is impossible to obtain quite con- 
cordant results in drying material like grain, which will continue to lose 
water in the drying oven at 100" C. for an indefinite period. 

Fig. 4 shows the relationship of green to dry weight — all three 
samples in 1907 follow a very parallel course, the notable features of 



_ - < r . r- " 

5^ 



Plot 10 
R.F. 
Plot 3 



3 6 9 12 15 18 21 24 27 30 33 36 39 clays 

Fig. 4. °/ dry weight to green weight, 1907 only. 

which are a change of curvature after the third period, and another 
change about six or nine days before cutting. Both these breaks are 
symptomatic ; as will be seen later the first marks the final contraction 
and drying up of the pericarp, the second indicates the beginning of 
desiccation and the conclusion of the migration. 

Fig. 5 shows the actual water contained in 1000 grains and is highly 
instructive : the water rises until the third or fourth period, then 
it remains approximately constant in amount until six days from 
cutting, after which it falls rapidly. Again the two critical dates 
are about twelve days after the first sampling and six days before 
cutting. 

Nitrogen. The percentage of nitrogen in the dry matter of the 
grain (Fig. 6) falls rapidly at first but after the first six periods becomes 



202 The Development of the Grain of Wheat 


































































-^ 


<^ 


----- 


TTTT- 


: ^> 




J 


§S 










y 




— - 


— — 





__-:"\/ 












s 






















// s 


' 






















fA'' 















































































Plot 3 
R.F. 



3 6 9 12 15 18 21 24 27 30 33 36 39 days 

Fig. 5. Actual water contained in 1000 grains, 1907 and 1908. 



275 


































2 50 




s 


































-*-,, 




























225 


\i. 




































> 






























\\ 




'• : v 


























2-00 












































: - 


x 




















1 75 
V50 

1-25 
100 














*=^ 


~ " 


X 














^ 


\ 






~x~n^_ 






^ „ v 


-ii 


— " r 


■-•-._ 


^_ 


^^ 
























































































75 
•50 
•25 





















































































R.F. 
Plot 1Q 



Plot 3 
1908 



3 6 9 12 15 18 21 24 27 30 33 36 39 days' 

Fig. 6. °/ nitrogen in dry matter of grain, 1907 and 1908. The dark line shows the 
mean curve of the three plots for 1907, and is placed three squares too low for 
the sake of clearness. 



W. E. Brenchley and A. D. Hall 



203 



fairly constant : there is some indication of a rise towards the end, but 
the curves are not smooth enough to be sure of this, though as will be 
seen later it is explicable by the continued loss of non-nitrogenous 
matter by respiration. The actual nitrogen in 1000 grains (Fig. 7) 



grams 
9 







































































y 


a 


/ 
/ 


-/■- 


^ 


















/ 


C-' 




















^ 


„."■' 


* 








































-^ 


^' 


' 


















j-j2 


€^' 


"'" 

















































Plot 10 
R.F. 
Plot 3 



12 15 18 21 24 27 30 33 36 39 days 

Fig. 7. Actual nitrogen contained in 1000 grains, 1907 and 1908. 

rises regularly until the last three-day period. The very steady incre- 
ment of nitrogen in itself disposes of the opinion that the nitrogenous 
constituents enter the endosperm first, and that the later filling of the 
grain consists mainly of starch. Confirmation is obtained by recalcu- 
lating the results so as to ascertain the proportion of nitrogen in the 
dry matter that has entered the grain between successive dates, though 
the figures obtained can only be viewed very generally, because the 
experimental errors are accumulated in quantities that are not them- 
selves large. Comparing, however, the first and second halves of the 
whole period we get the following proportions of nitrogen in the dry 
matter. 



Percentages of Nitrogen in Dry Matter entering the Grain. 

Plot 3. 

July 19— August 6 1'667 

August 6— 24 1-681 



14—5 



204 The Development of the Grain of Wheat 

Plot 10. 

July 16— August 3 1*698 

August 3— 21 1-868 

Bed Fife, 1.907. 

July 25— August 12 1-692 

August 12—30 2452 

Square Head's Master, 1908. 

July 3—21 1552 

July 21— August 8 1'912 

These figures show that the material filled into the grain is more 
nitrogenous in the later than in the earlier stages. 

A better idea of what takes place may be obtained by dividing the 
whole period into three stages suggested by the variations in the water 
in the grain. 





Plot 3 


Plot 10 


Eed Fife 


Square Head's 
Master, 1908 


Stage of increasing water ... 

Stage of constant water 

Stage of desiccation 


1-932 

1-592 

? 


1-916 
1-631 
2-700 


2-035 
1-930 
2-415 


1-612 
1-677 
1-989 





In the first stage the larger part of the grain consists of the soft 
tissue forming the pericarp, the subsequent shrinkage of which into dry 
membranes is practically complete by the end of the first stage. The 
endosperm exists during all the first stage and at the end is beginning 
to show starch, &c. throughout. The material forming the pericarp 
evidently contains more nitrogen than that which enters the endo- 
sperm later. The middle stage is characterised by the filling in of the 
endosperm, and in the last stage the migration is coming to an end ; 
during this period the material that is stored appears to be more 
nitrogenous because the entry is slow, while the losses by respiration, 
which fall wholly on the non-nitrogenous substance, are still going on. 

Ash and Phosphoric Acid. The proportion of ash in dry matter, 
and the amounts of ash and of phosphoric acid in 1000 grains, yield 
curves exactly similar to those given by nitrogen, indicating that the ash 
and the phosphoric acid enter the grain pari passu with the nitrogen. 
Table I. shows the ratio of phosphoric acid to nitrogen for each sample, 
and indicates that the wheat on each plot manufactures material 
possessing a composition special to itself, but one which remains 



W. E. Brenchley and A. D. Hall 



205 



approximately constant during the whole formation of the grain. Similar 
constant ratios are obtained between the nitrogen, phosphoric acid and 
carbohydrates, the carbohydrates being reckoned as dry matter less 
protein and ash. 

Nitrogen 



Table I. 



Phosphoric acid 



Ratio. 



Days 


Plot 3 


Plot 10 


Red Fife 


Square Head's 
Master, 1908 





2-150 






2-170 


3 


2-154 


— 


— 


1-945 


6 


2-122 


2-351 


— 


1-950 


9 


1-995 


2-323 


1-907 


1-839 


12 


2-051 


2-253 


1-877 


1-717 


15 


2-220 


2-528 


1-909 


1-687 


18 


1-869 


2-315 


1-850 


1-690 


21 


2-035 


2-268 


1-819 


1-643 


24 


1-890 


2-503 


2-000 


1-848 


27 


1-859 


2-453 


1-805 


1-843 


30 


2-279 


2-409 


1-821 


1-786 


33 


1-963 


2-167 


1-909 


1-826 


36 


2-064 


2-294 


1-851 


1-811 


39 


1-987 


2-375 


1-977 





4-0 




























32 


\\ 




























\ 




























\ 




2 


~"~K^ 




























~~ 


'"-- 


"^Sj 




5^ 









-.—- 


2-0 


























~~~- 



































































•8 




























•4 





























R.F. 
1908 
Plot 3 
Plot 10 



Fig. 8. °/ ash in dry matter, 1907 and 1908. 
It may be noted here that when wheats from different plots, &c. are 
compared, there is no connexion between the actual percentage of 



206 The Development of the Grain of Wheat 

nitrogen in the grain and the nitrogen-phosphoric acid ratio. It has 
often been supposed that the extent to which the plant can utilise 
nitroo-en in the soil is dependent upon the phosphoric acid also present, 
because the phosphoric acid acts in some way as a carrier of nitrogen, 

grams 
10 















































































^7^ 
























/ m ,gS 


•*~~ 
























,..-> 


r 1 ' 




















^, 


s\ , -~' 


,'' 
























'' 
























' ^ 


/ 






















y - 


>' 
























y-y 


-''" 
























■'' 


■'' 




















^ 


/' 



















































jrams 
•50 



Plot 3 
R.F. 
Plot 10 



6 9 12 15 18 21 24 27 30 33 36 

Fig. 9. Actual ash in 1000 grains, 1907 and 1908. 



39 days 



































































































^.- 


„.</ 




__ 














/ 




/ 




*'' 


















^ 


- — 


/ 


'' 
















^ 


A? 


"" 




















^^ 


"-'-'' 












































** 



























R.F. 
Plot 3 



6 9 12 15 18 21 24 27 30 33 36 39 days 

Fig. 10. Actual P 2 5 in 1000 grains, 1907 and 1908. 



W. E. Brenchley and A. D. Hall 



207 



but the wheat from plot 10 is exceptionally nitrogenous for the variety, 
and at the same time exceptionally poor in phosphoric acid. 

Other determinations. A certain number of determinations were 
made in order to asertain if any marked change could be found in the 
nature of the materials accumulated iu the grain, by which the degree 
of ripeness could be gauged. Fig. 11 shows the percentage of sugar 

































„- 


V * 




























\\ \ 




























\\\ 
























12 




\\ 


( 
























\ 


\\ 


























' 


V 






















10 






\ ^ 


























\ ^ v 






























\"~~- 


t 


























^\ 


\ 


























\ 




--, 




























\\ 


\ 


























^ \ 


\ 


























\ 




v^_ 


„ 







• 
































n 





























Plot 10 
Plot 3 
R.F. 



Fig. 11. 



12 15 18 21 24 27 30 33 36 

% dextrose in dry matter of grain, 1907. 



(dextrose) in the dry matter of the grain : it is high at first, about 
15 per cent., but falls rapidly to about 2 per cent., at which figure it 
keeps fairly constant for the last fortnight before cutting. On recal- 
culating to ascertain the amount of dextrose per 1000 grains (Fig. 12) 



































y 




Sr"' 


k 
















,'' 


^x 


















> 




/ 

/ 
/ 






N 


-•"'" 




\\ 


^ 


i 






/ 


\ 


' 












*""~- 


>.N 


/ 




-.^ 


i 

























































Plot 3 
R.F 



9 12 15 18 21 24 27 30 33 36 39 days 

Fig. 12. Actual dextrose in 1000 grains, 1907. 



208 The Development of the Grain of Wheat 

it is seen to increase for the first three or four periods (i.e. while the 
living tissues of the pericarp form the most prominent feature in the 
grain), then it falls rapidly, and during the last fortnight it remains 
approximately constant, though the figures are evidently affected by a 
large experimental error. 



























































700 








/ / 




















600 
500 





/ 








\\ 


\ 




/ 


\ 








1 
/ 




1 






-V 




w 


\ 
\ 
\ 










/ 


1 ,' 




: / 








s 




\ 




,..* 




300 


, 


/ 


• 
















\\ 


.-'"' 




\ 

X 


100 























































Plot3 
Plot 10 



39 days 



Fig. 13. Maltose produced per 100 of dry matter, 1907. The dark line shows the mean 
curve for the three plots, placed three squares too low for the sake of clearness. 

Determinations of the diastatic power were made by rapidly 
macerating the fresh grain and adding it to starch paste: Fig. 13 
shows the amount of maltose thus produced per 100 of dry matter in 
the grain. The results are subject to a large experimental error, but 
indicate that the diastatic power of the material, taken as a whole, 
rises during the first four or five periods and then falls steadily. 
Again recalculating the results to show diastatic power per 1000 grains 
(Fig. 14), this property rises for five periods and then probably remains 
constant. 

Owing to an accident only one set of determinations of protein 
nitrogen are available, for 1908 ; these show (Fig. 15) a marked rise in 
the proportion of nitrogen in the protein form during the period of 
experiment. At first about 72 per cent, of the nitrogen is combined as 
protein but this gradually rises to over 99 per cent. 

On the same figure is shown the actual amount of non-protein 
nitrogen contained in 1000 grains ; it rises at first, then remains 
approximately constant, and finally falls rapidly during the last desic- 



W. E. Brenchley and A. D. Hall 



209 



cation stage. Evidently the end process of ripening is accompanied by 
a change from non-protein to protein nitrogenous compounds. 

General outline of the process of migration. It is now possible to 
summarise the whole process of the migration of the reserve materials 
into the wheat grain. The first samples were taken about ten days after 
flowering ; at this time the endosperm is just formed, but the grain is 
in the main made up of the active living tissues constituting the pericarp. 
The figures for July 14th in Plate XV (taken from W. E. Brenchley, 
loc. cit.) show the structure of the grain at this stage. During the 



grams 
220 













































i 


\ 
























i 
/ 


\ 
\ 

\ 
















/ /. 


\ 


\. 


-*-V 


/ \ 
/' \ 


\ 


7\ 












/,/ 


/ 


\ 
\ 


/ > 
/ 


/"""> 


i 


N v \ 


/ 






































,' 






















s S* 




,' / 




















*■' 















































































Plot 3 
Plot 10 



39 days 



Fig. 14. Maltose produced per 1000 grains, 1907. The dark line shows the mean curve 
of the three plots, placed two squares too low for the sake of clearness. 

next twelve days the endosperm is beginning to fill, as shown by the 
appearance of starch grains in the cells, until by the end of the period 
starch is to be found throughout the endosperm. But the most 
characteristic feature of this stage is the depletion of the cells in the 
pericarp and their crushing together, until they become nothing more 
than membranes containing no living cells ; the end of this stage being 
shown by the second set of figures in the plate. It is during this 
period that the nitrogen percentage of the grain is falling rapidly ; the 
cells of the pericarp when active evidently possess a comparatively high 



210 The Development of the Gram of Wheat 

proportion of nitrogen and ash, though the percentage of phosphoric acid 
in the ash is low. Both the dextrose and the diastatic power of the 
grain are rising during this period. 



too 




























































80 
70 
60 


































































































40 

30 








































































\ 


20 

10 


























\ 
























' 



grams 
06 



3 6 9 12 15 18 21 24 27 30 33 36 days 

Fig. 15. °/ protein nitrogen in total nitrogen, 1908 (upper curve). Actual 
non-protein nitrogen in 1000 grains (lower curve). 

During the next period, which lasts about a month, the endosperm is 
being filled up, and the dry weight of the grain is more than trebled, 
but the actual amount of water present in the grain remains approxi- 
mately constant. Throughout this time the material moved by the 
plant and stored in the endosperm appears to be of constant composition, 
as indicated by the uniformity of the N: P 2 5 : carbohydrate ratio of the 
material entering between successive dates. Each wheat however 
elaborates and stores a characteristic material, the composition of which 
is determined beforehand by variety, soil (including manure), climate, 
and similar factors independent of the migration process. The microscopic 
examination of the grain would show that the cells of the endosperm are 
filled progressively beginning from the base of the grain and proceeding 
towards the tip, or end at which the embryo is developed, each set of 
cells being successively filled up and then as it were put out of action. 
The fact that the total amount of water, non-protein nitrogen, diastatic 
power, and dextrose (though this latter material does not become constant 



W. E. Brenchlby and A. D. Hall 211 

until a later period than the others) remain constant during the rilling 
stage, indicates that these materials belong to the active cells which 
are being rilled, rather than to the cells which have been rilled up and 
put out of action. 

Finally ripening begins about six days before cutting, and the 
characteristic feature is the rapid desiccation of the grain ; the actual 
water falls as the remaining active cells fill up, the non-protein nitrogen 
drops, and the precentage of nitrogen in the material still entering 
increases, because the losses by respiration overtake the gain by 
migration. The maximum weight of dry matter is reached a few 
days before the grain appears to be ripe for cutting, because the intake 
ceases, while respiration still continues. Cytologically this last stage of 
ripening is marked by the progressive destruction of the nuclei in the 
endosperm as they are squeezed into networks by the pressure of the 
starch grains, but no sequence can be traced in the regions showing 
such deformed nuclei, such as was observed by Brown and Escombe 
in barley, which shows a progressive ' nuclear senescence ' with 
ripening. 

Relation of the migration process to the nutrition of the whole 
plant. Since the publication of Pierre's investigations (loc. cit.) it has 
been generally held that the wheat plant ceases to draw nutriment from 
the soil after a comparatively early date — the flowering period or a little 
later. Assimilation, however, was considered to go on later, but to cease 
in its turn before the migration into the grain had been completed ; 
it has even been held that there is a return of nutrient materials to 
the soil, an actual excretion of phosphoric acid, nitrogen, &c. in the 
final stages. Such a complete cessation of nutrition and assimilation 
must however be a matter of season and climate ; as long as any part 
of the plant remains green assimilation will go on, water will be drawn 
from the soil, and with the transpiration current nutrient materials will 
enter the plant. In 1908 the straw belonging to each of the marked 
ears was cut off close to the ground and analysed in order to trace the 
relationship between migration and the nutrition of the whole plant. 
The ratio between grain and straw in these selected shoots was deter- 
mined and as before the unit yield at each date is represented by the 
material contained in 1000 grains and also in the straw which was found 
to be associated with 1000 grains at that period. 

Fig. 16 shows the dry matter curves for the whole plant and for the 
grain ; from which it will be seen that the dry weight of the whole plant 
increases up to within a week of cutting, i.e. the point when desiccation 



212 The Development of the Grain of Wheat 

in the grain sets in. It is evident that assimilation does not cease until 
migration is nearly complete. Respiration continues later still, because 
the weight of the whole plant falls in the last week. 

Fig. 17 shows the nitrogen in the whole plant and in the grain ; here 
again, though the curve is not very smooth, there is no evidence of any 
cessation in the intake of nitrogen until within a few days of the date 
of cutting. 



120 


















































no 


























100 


— 
























90 

80 
70 

CO 










































































50 
40 
30 










































































20 
10 

n 











































































21 24 27 30 33 36 days 



Fig. 16. Dry weights of whole plant and grain, 1908 (whole plant = weight of 
1000 grains + weight of straw calculated as equivalent to 1000 grains). 



Fig. 18 shows the ash in the whole plant and in the grain ; similarly 
it is seen that the intake of ash by the plant, though not so pronounced 
during the period under review, continues to within a week of cutting. 
The amount of ash then becomes stationary, the slight fall indicated in 
the last week being within the limits of experimental error. Exactly 
similar conclusions are to be drawn from the phosphoric acid curves set 
out in Fig. 19. 



W. E. Brenchlby and A. D. Hall 



213 













































































/ 




























































/ 


/ 






















/ 



































































































































3 6 9 12 15 18 "21 24 27 30 33 36 days 

Fig. 17. Nitrogen in whole plant and in grain, 1908. 



7 

4 

3 

2 

1 



12 15 18 21 24 27 30 33 36 days 

Fig. 18. Ash in whole plant and in grain, 1908. 



214 The Development of the Grain of Wheat 

Fig. 20, showing the percentage of nitrogen and phosphoric acid in 
the straw, has been drawn in order to demonstrate how the feeding value 
of the straw declines as the grain forms. 



6 
5 

4 

9 

1 



6 9 12 15 18 21 24 27 30 33 36 days 

Fig. 19. P 2 D in whole plant and in grain, 1908. 



8 

6 — ^^ 

2 — 

1 

I I 1 1 



N 
Pa°5 



Fig. 20. % nitrogen and P„0 5 in dry matter of straw, 1908. 

It should of course be remembered that in these results no account 
is taken of the root of the plant, which cannot be removed from the soil 
without both loss of the fine roots and the introduction of foreign 
matter ; migration will no doubt take place from the root to the seed, 
but the weight of root bears too small a proportion to that of the 



JOURNAL OF AGRICULTURAL SCIENCE. Vol. III. No. 2. 



PLATE XV 



July U^ July 23?$ Aug.lOty 




-pc. 



-end. 



-end. 






W. E. Brbnchley and A. D. Hall 215 

whole plant to account for the rise in dry matter, &c. that is observed in 
the grain and straw during the migration period. The question of 
when nutrition and assimilation finally cease can only be definitely 
settled when the roots also can be examined, and experiments to that 
end are now in progress. Meantime the evidence derived from our 
experiments is against the view that either nutrition or assimilation 
ceases before the final ripening off of the wheat grain ; this, however, 
may only be true for the comparatively humid English climate where 
the wheat plant retains some green active tissue until harvest is close 
at hand. 

Summary. 

A study during 1907 and 1908 of various plots of wheat cut at three- 
day intervals leads to the following general conclusions : 

(1) The whole plant, and with it the nitrogen, ash, and phosphoric 
acid it contains, increases in weight until about a week before it would 
be regarded as ready to cut. Some decrease of dry weight takes place 
during the last week. 

(2) In the formation of the grain three stages may be distinguished : 
(a) a period during which the pericarp is the most prominent 

feature, 
(6) the main period during which the endosperm is filled, 
(c) the ripening period characterised by the desiccation of the 

grain. 

(3) For the filling of the endosperm each plant possesses as it were 
a special mould, and continually moves into the grain uniform material 
cast in that mould, possessing always the same ratio of nitrogenous to 
non-nitrogenous materials and ash. The character of the mould 
possessed by each plant is determined by variety, soil, season, &c. 

(4) The main feature of the ripening process is desiccation rather 
than the setting in of such chemical changes as the conversion of sugars 
into starch, non-protein into protein, though the latter change also takes 
place. 

(5) The maximum dry weight of grain is attained a day or two 
before the grain would be regarded as ripe by the farmer. Allowing for 
the fact that the tillered shoots are a little behind the central shoots, 
no loss of weight in the crop will be incurred by cutting before the 
corn appears quite ripe, while a number of accidental mechanical losses 
due to birds, shedding, weather, may thus be avoided. Other experi- 
ments have shown that, though there may be no gain, there will be no 
loss in the quality of the wheat due to such early cutting. 



216 The Development of the Grain of Wheat 



Appendix I. 
Broadbalk, Plot 3, 1907. 



















Maltose 


Date 


Green 
weight 


Dry 

weight 


Specific 


°/o 

nitrogen 


7o ash 
in dry 
matter 


7oPA 


7o 
dextrose 


produced 
per 100 
of dry 

matter 


of 1000 
grains 


of 1000 
grains 


gravity 


in dry 
matter 


in ash 


in dry 

matter 




grams 


grams 














July 16 


13-75 


3-51 


1116 


2-679 


3-70 


33-66 


— 


— 


„ 19 


21-05 


5-43 


1-116 


2-406 


303 


36-91 


14-99 


339-4 


,, 22 


32-47 


8-14 


1-113 


2-458 


3-14 


36-88 


11-08 


324-7 


„ 25 


39-70 


11-16 


1-116 


2-167 


2-80 


38-73 


7-36 


541-4 


„ 28 


45-95 


14-05 


1-099 


2-119 


2-66 


38-86 


6-71 


650-7 


„ 31 


51-30 


17-99 


1-116 


2 055 


2-39 


38-68 


6-23 


597-1 


Aug. 3 


56-69 


21-15 


1-128 


1-856 


2-38 


40-35 


3-70 


510-6 


„ 6 


57-91 


24-97 


1-113 


1-828 


2-16 


42-54 


2-42 


442-9 


,. 9 


62-48 


28-98 


1-196 


1-801 


2-16 


44-17 


2-17 


412-0 


„ 12 


63-68 


32-20 


1-215 


1-720 


2-09 


44-30 


1-86 


378-1 


„ 15 


6319 


35-09 


1-218 


1-856 


1-89 


44-06 


1-46 


277-9 


„ 18 


70-89 


37 93 


1-231 


1-787 


1-96 


46-50 


1-99 


441-6 


» 21 


66-30 


38-69 


1-204 


1-846 


1-94 


46-13 


1-91 


343-7 


„ 24 


61-01 


37-96 


1-271 


1-778 


1-93 


46-30 


2-02 


322-1 



July 


13 


>> 


If, 




11) 




'!■> 




25 




28 




31 


Aug. 


3 


>» 


6 


>> 


9 


>> 


12 


) * 


15 




18 




21 



July 22 

„ 25 

„ 28 

„ 31 

Aug. 3 

„ 6 

„ 9 

., 12 

„ 15 

„ 18 

„ 21 

„ 24 

„ 27 

,, 30 





Broadbalk 


, Plot 


10, 1907. 




12-45 


2-93 


1-169 


2-910 


— 


— 


— 


20-92 


5-36 


1-136 


2-694 


— 


— 


16-76 


30-81 


8-13 


1-122 


2-611 


2-87 


38-64 


12-42 


40-21 


1116 


1-114 


2-445 


2-80 


37-62 


9-24 


43-59 


13-69 


1-098 


2-128 


2-43 


38-91 


8-73 


50-19 


16-95 


1-119 


2-100 


2-28 


39-11 


6-41 


51-85 


20 34 


1-125 


2-113 


2-18 


41-72 


5-84 


55 70 


23 66 


1-120 


1-923 


2-01 


42-24 


2-97 


60-43 


28-23 


1-153 


1-845 


1-84 


40-07 


1-66 


59-99 


30-10 


1-208 


1-877 


1-81 


42-22 


2-18 


64-47 


34-45 


1-251 


1-832 


1-85 


41-08 


1-38 


65 65 


34-45 


1-221 


1-829 


1-96 


43-18 


1-17 


65-29 


37-93 


1-236 


1-875 


1-84 


44-35 


2-48 


56-93 


37-65 


1-249 


1-903 


1-86 


43-19 


— 



Red Fife, 1907. 



15-66 


3-89 


1-110 


2-552 


— 


38-42 


13-98 


19-64 


5-17 


1-098 


2-322 


— 


40-13 


14-93 


25-17 


6-79 


1-085 


2-404 


— 


42-59 


11-98 


31-54 


9-58 


1112 


2-271 


2-839 


41-94 


9-13 


38-20 


12-01 


1-088 


2-202 


2-749 


42-69 


6-00 


41-86 


15-17 


1-124 


2-049 


2-422 


44-29 


5-17 


46-05 


18-18 


1-157 


1-967 


2-291 


46-41 


3-21 


50-55 


22-49 


1-185 


1-837 


2-201 


45-87 


2-27 


52-47 


24-63 


1-184 


1-848 


2-151 


46-04 


1-73 


56-73 


28-68 


1-197 


1-886 


2-075 


50-34 


2-75 


57-52 


30-75 


1-187 


1-879 


2-046 


50-43 


2-60 


59-40 


32-61 


1-231 


2-030 


2-075 


51-26 


2-39 


59-33 


33-58 


1-238 


1-979 


2-117 


50-52 


2-32 


54-48 


34-33 


1-254 


2-050 


2-133 


48-62 


1-82 



248-6 
404-7 
235-7 
701-4 
694-1 
597-0 
376-1 
430-7 
332-4 
242-2 
294-8 
327-6 



230-9 
358-9 
556-3 
563-9 
705-2 
735-9 
440-5 
451-1 
372-2 
529-3 
394-3 
316-6 
307-1 
218-5 



W. E. Brenchley and A. D. Hall 

Square Head's Master, 1908. 



217 



Date 


Green 

weight 
of 1000 


Dry 
weight 
of 1000 


>0 

nitrogen 
in dry 
matter. 


°/o. Pro- 
tein N. 
in total 
nitrogen. 


°l 

nitrogen 

in dry 

matter. 


°/ ash in dry 
matter 


7o P 2 5 in 

ash 


Grain to 
Straw 
( = 100) 

ratio dry 












grains 


grains 


Grain 


Grain 


Straw 


Grain 


Straw 


Grain 


Straw 


weights 




grams 


grams 


















July 3 


15-13 


3-81 


2-676 


72 13 


•731 


3-42 


5-00 


36-08 


8-80 


4-24 


„ 6 


24-78 


6-55 


2-245 


77-50 


•684 


3-04 


4-89 


38-03 


9-26 


7-16 


„ 9 


35-82 


9-73 


2-177 


81-79 


•736 


2-64 


5-18 


42-22 


8-29 


10-40 


„ 12 


46-51 


13-44 


1-926 


78-09 


•699 


2-46 


5-24 


42-62 


8-24 


15-50 


„ 15 


53-20 


17-26 


1-846 


83-48 


•663 


2-53 


5-48 


42-54 


7-26 


19-41 


„ 18 


59-74 


21-13 


1-737 


87-12 


•642 


2-37 


5-38 


43-40 


6-92 


24-56 


„ 21 


63-41 


24-49 


1-727 


86-36 


•599 


2-28 


5-57 


44-73 


5-73 


28-90 


„ 24 


68-61 


29-67 


1-643 


89-73 


•488 


2 16 


5-79 


46-33 


5-24 


35-91 


„ 27 


75-54 


35-24 


1-760 


91-91 


•505 


2-09 


6-35 


45-54 


4-83 


44-63 


„ 30 


79-38 


40-06 


1-733 


91-27 


•420 


1-96 


6-55 


47-91 


4-43 


52-46 


Aug. 2 


82-79 


43-73 


1-754 


96-42 


•343 


1-96 


6-91 


50-11 


4-18 


57-92 


„ 5 


81-26 


45-88 


1-801 


96-72 


•356 


1-95 


7-28 


50-50 


4-25 


63-54 


„ 8 


75-61 


45-83 


1-813 


99-13 


•348 


2-00 


7-19 


50-18 


4-07 


66-03 



